Subconfluent Cells Research Articles

Effect of transforming growth factor‐beta1 on expression of the connective tissue growth factor (CCN2/CTGF) gene in normal human gingival fibroblasts and periodontal ligament cells

Connective tissue growth factor (CCN2/CTGF) plays an important role in wound healing and regulation of the extracellular matrix in periodontal tissue. However, the functional relationship between altered transforming growth factor-beta1 levels and CCN2/CTGF has not been extensively investigated in human gingival fibroblasts and periodontal ligament cells. This study investigated the effects of transforming growth factor-beta1 on the expression of the CCN2/CTGF gene in human gingival fibroblasts and periodontal ligament cells in vitro. Cells were isolated from normal periodontal tissues and cultured in Dulbecco's modified Eagle's minimal essential medium/F12 containing 10% fetal bovine serum. Subconfluent cells were maintained under serum deprivation for 24 h then treated with Dulbecco's modified Eagle's minimal essential medium/F12 containing 0.5% fetal bovine serum (control) and 0.1, 1, 5 or 10 ng/mL of transforming growth factor-beta1 for 24, 48 or 72 h. The effects of transforming growth factor-beta1 on CCN2/CTGF mRNA expression were measured by reverse transcription-polymerase chain reaction. CCN2/CTGF protein was quantitatively analyzed using enzyme-liked immunosorbent assay. Subcellular distribution of CCN2/CTGF protein in both human gingival fibroblasts and periodontal ligament cells was observed using immunofluorescence microscopy. In both human gingival fibroblasts and periodontal ligament cells, the expression of CCN2/CTGF mRNA and CCN2/CTGF protein was significantly increased, in a dose- and time-dependent manner, in the presence of transforming growth factor-beta1. Moreover, immunofluorescence analysis indicated that immunoreactivity to CCN2/CTGF showed a granular pattern of protein localization. The expression of CCN2/CTGF mRNA and protein was induced by transforming growth factor-beta1 in human gingival fibroblasts and periodontal ligament cells. These results suggest that CCN2/CTGF plays an important role in wound healing and in the regeneration of periodontal tissue.

Negative and Positive Regulation of MAPK Phosphatase 3 Controls Platelet-derived Growth Factor-induced Erk Activation

MAPK phosphatases (MKPs) are dual specificity phosphatases that dephosphorylate and thereby inactivate MAPKs. In the present study, we provide evidence that platelet-derived growth factor BB (PDGF-BB) regulates MKP3 (DUSP6), which is considered to be a phosphatase highly selective for Erk. Intriguingly, we observed that Mek is positively regulated by MKP3, whereas Erk itself is negatively regulated. In addition, we found that activation of PDGF receptor alpha or beta leads to a rapid proteasomal degradation of MKP3 in a manner that requires Mek activation; this feed-forward mechanism was found to be essential for efficient Erk phosphorylation. We could also demonstrate that PDGF-BB stimulation induces phosphorylation of MKP3 at Ser-174 and Ser-300; phosphorylation of Ser-174 is involved in PDGF-induced MKP3 degradation, since mutation of this site stabilized MKP3. Moreover, activated Erk induces mkp3 expression, leading to restoration of MKP3 levels after 1-2 h and a concomitant dephosphorylation of Erk in cells with activated PDGFRalpha. Reducing the MKP3 level by small interfering RNA leads to an increased Erk activation and mitogenic response to PDGF-BB. In conclusion, MKP3 is an important regulator of PDGF-induced Erk phosphorylation acting in both a rapid positive feed-forward and a later negative feed-back loop.

Ribosomal S6 Kinase 2 Directly Phosphorylates the 5-Hydroxytryptamine 2A (5-HT2A) Serotonin Receptor, Thereby Modulating 5-HT2A Signaling

The 5-hydroxytryptamine 2A (5-HT(2A)) receptor is a member of the G protein-coupled receptor superfamily (GPCR) and plays a key role in transducing a variety of cellular signals elicited by 5-hydroxytryptamine in both peripheral and central tissues. Despite its broad physiological importance, our current understanding of 5-HT(2A) receptor regulation is incomplete. We recently reported the novel finding that the multifunctional ERK effector ribosomal S6 kinase 2 (RSK2) physically interacts with the 5-HT(2A) receptor third intracellular (i3) loop and modulates receptor signaling (Sheffler, D. J., Kroeze, W. K., Garcia, B. G., Deutch, A. Y., Hufeisen, S. J., Leahy, P., Bruning, J. C., and Roth, B. L. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 4717-4722). We report here that RSK2 directly phosphorylates the 5-HT(2A) receptor i3 loop at the conserved residue Ser-314, thereby modulating 5-HT(2A) receptor signaling. Furthermore, these studies led to the discovery that RSK2 is required for epidermal growth factor-mediated heterologous desensitization of the 5-HT(2A) receptor. We arrived at these conclusions via multiple lines of evidence, including in vitro kinase experiments, tandem mass spectrometry, and site-directed mutagenesis. Our findings were further validated using phospho-specific Western blot analysis, metabolic labeling studies, and whole-cell signaling experiments. These results support a novel regulatory mechanism in which a downstream effector of the ERK/MAPK pathway directly interacts with, phosphorylates, and modulates signaling of the 5-HT(2A) serotonin receptor. To our knowledge, these findings are the first to demonstrate that a downstream member of the ERK/MAPK cascade phosphorylates a GPCR as well as mediates cross-talk between a growth factor and a GPCR.

The Anaphase Promoting Complex Induces Substrate Degradation during Neuronal Differentiation

The anaphase promoting complex (APC) is an E3 ubiquitin ligase required for the metaphase-to-anaphase transition and mitotic exit. However, APC also plays roles in G(1), where it is regulated by Cdh1, and APC activity has also been detected in differentiated and non-proliferating cells, suggesting that it may play roles outside the cell cycle. Here, we report that disrupting APC(Cdh1) activity inhibits neurite outgrowth of both PC12 pheochromocytoma cells and primary cerebellar granule cells. APC(Cdh1) activity dramatically increases as PC12 cells differentiate in response to nerve growth factor. Furthermore, a key target degraded by APC(Cdh1) following nerve growth factor treatment is the F-box protein Skp2, and APC(Cdh1)-mediated destruction of Skp2 is essential for proper terminal differentiation of neuronal precursors.

Induction of protoporphyrin IX by aminolaevulinic acid in actinic keratosis, psoriasis and normal skin: preferential porphyrin enrichment in differentiated cells

In photodynamic therapy the endogenous photosensitizer protoporphyrin IX (PpIX) is synthesized following topical application of aminolaevulinic acid (ALA). However, different tissues have distinct PpIX-accumulating properties, due to differences in penetration of ALA through the stratum corneum and/or alterations in the haem cycle. Preferential PpIX accumulation has been reported in terminally differentiated cell cultures but ex vivo data are lacking. To study the intrinsic PpIX-accumulating capabilities of skin explants from lesional and nonlesional skin in psoriasis and actinic keratosis, with normal skin serving as a control, and to study PpIX accumulation in relation to differentiation status in normal skin. Skin explants from patients with psoriasis, actinic keratosis and normal skin were incubated with ALA and PpIX was measured spectrofluorometrically. PpIX was measured in basal (beta1-integrin-positive) and suprabasal (beta1-integrin-negative) keratinocytes in normal skin. In addition, PpIX accumulation was studied in cell cultures at different levels of confluence and after induction of terminal differentiation. No significant differences in PpIX content were found between the different tissues. However, increased PpIX was found in beta1-integrin-negative compared with beta1-integrin-positive cells. In addition, in subconfluent cell cultures less PpIX was found compared with confluent cell cultures. Induction of terminal differentiation in vitro, however, resulted in less PpIX, which was likely to be related to cell volume. As no significant differences in PpIX synthesis could be found between the different tissue types, these data emphasize the importance of the penetration route of ALA through the stratum corneum. Preferential PpIX accumulation observed in suprabasal epidermal keratinocytes and confluent cell cultures points towards a terminal differentiation-specific effect.

DOCK5 and DOCK1 Regulate Caco-2 Intestinal Epithelial Cell Spreading and Migration on Collagen IV

We observed previously that combined small interfering RNAs (siRNAs) targeting CrkII and CrkL, known activators of guanine nucleotide exchange factor DOCK1, strongly inhibit Caco-2 intestinal epithelial cell spreading and migration on collagen IV. DOCK1 siRNA reduced its expression >95% in Caco-2 cells but inhibited spreading much less than combined CrkII/CrkL siRNAs, suggesting that CrkII/CrkL interact with additional DOCK proteins. siRNA targeting DOCK5, a closely related DOCK1 family member, inhibited Caco-2 spreading similarly to DOCK1 siRNA, and the combined siRNAs synergistically inhibited spreading. Similar results were observed in human umbilical vein endothelial cells, and reverse transcriptase PCR demonstrated DOCK5 siRNA reduction of DOCK5 expression in both cell types. Combined DOCK1/DOCK5 siRNAs also inhibited Caco-2 migration and lamellipodial extension. Expression of DOCK5 cDNA, with silent mutations in the siRNA target region allowing expression simultaneously with DOCK5 siRNA, required CrkII/CrkL to restore cell spreading and DOCK5 coimmunoprecipitated with CrkII and CrkL. DOCK5 association with CrkII and CrkL was greatly reduced by mutations in their NH2-terminal SH3 domains. Expression of the DOCK5 COOH-terminal region (Met1738-Gln1870), containing potential Src homology 3 domain-binding proline-rich sites but lacking other functional regions, inhibited Caco-2 spreading and coimmunoprecipitated with CrkL. Coimmunoprecipitation of full-length DOCK5 with CrkL was strongly reduced by deletion of DOCK5 COOH-terminal amino acids 1832-1870. Green fluorescent protein-tagged DOCK5 localized to the membrane of Caco-2 cells spreading on collagen IV. In these studies, we describe human DOCK5 cloning and expression, our results indicating that, along with DOCK1, DOCK5 is an important mediator of CrkII/CrkL regulation of Caco-2 spreading and migration on collagen IV.

Adherens junction-dependent PI3K/Akt activation induces resistance to genotoxin-induced cell death in differentiated intestinal epithelial cells

The crypt–villi axis of intestinal mucosa maintains homeostasis by renewal of epithelia, and also exhibits different properties from undifferentiated to terminally differentiated cells. We investigated differential susceptibility to genotoxin-induced cell death, based on the degree of differentiation of epithelial cells, and its mechanism. Differentiation was induced by post-confluence culture in Caco-2 cells. Methyl methanesulfonate (MMS), a direct-acting DNA alkylating agent, was used for genotoxin-induced cell death. Compared to subconfluent Caco-2 cells, 7 days post-confluent cells showed resistance to MMS-induced cell death. With increasing expression of adherens junction components of E-cadherin and β-catenin, E-cadherin and p-Akt expression increased in 7 days post-confluent Caco-2 cells, and in human intestinal tissue, expression of E-cadherin and p-Akt also increased in the upper portion of villi, compared to the crypt. Inhibition of cell–cell adhesion using EGTA decreased Akt phosphorylation, which was reversed by calcium restoration. Akt phosphorylation by calcium-mediated cell–cell adhesion was more prominent in differentiated cells. In addition, treatment of a PI3K inhibitor, LY294002, inhibited Akt phosphorylation by calcium-mediated cell–cell adhesion. Finally, the differential sensitivity to MMS-induced cell death between subconfluent and 7 days post-confluent Caco-2 cells was eliminated by inhibiting cell–cell adhesion or PI3K. Our data demonstrated that cell adhesion-mediated PI3K/Akt activation could be one of the important mechanisms of resistance to genotoxin-induced cell death in differentiated epithelial cells.

Anti-angiogenic effects of non-peptide integrin αvβ3 specific antagonist on laser-induced choroidal neovascularization in mice

To evaluate the anti-angiogenic effects of integrin alphavbeta3 specific antagonist BS-1417 on laser-induced choroidal neovascularization (CNV) in mice. Male C57BL/6 mice were treated with daily intraperitoneal injections of BS-1417 or saline starting at the onset (day 0) of experiments. CNV was induced by laser photocoagulation the next day. Fluorescein angiograms (FA) and choroidal flatmount FITC-dextran perfusion were performed on experimental day 8. Histological and immunohistochemical examinations were performed with consecutive cryosections. Sub-confluent human vascular endothelial cells (HUVEC) were grown in vitro under various concentrations (0-10 microg/ml) of BS-1417 and the numbers of cell were measured at 48 hours of incubation. After fixation, immunocytochemistry was performed. BS-1417 significantly decreased the area of dye leakage determined by FA (40% of control, p = 0.0008) and reduced the CNV size found on choroidal flatmount (18% of control, p = 0.007). In histological findings, BS-1417 apparently suppressed the size of laser-induced CNV. Immunoreactivities for VEGF and integrin alphav were remarkably attenuated with BS-1417 compared to control. BS-1417 inhibited the growth and VEGF expression of HUVEC in vitro. The integrin alphavbeta3 may play a key role in the induction of laser-induced CNV. The antagonists for integrin alphavbeta3 may have therapeutic effects in CNV associated diseases such as age-related macular degenerations.

Image-based finite element modeling of alveolar epithelial cell injury during airway reopening

The acute respiratory distress syndrome (ARDS) is characterized by fluid accumulation in small pulmonary airways. The reopening of these fluid-filled airways involves the propagation of an air-liquid interface that exerts injurious hydrodynamic stresses on the epithelial cells (EpC) lining the airway walls. Previous experimental studies have demonstrated that these hydrodynamic stresses may cause rupture of the plasma membrane (i.e., cell necrosis) and have postulated that cell morphology plays a role in cell death. However, direct experimental measurement of stress and strain within the cell is intractable, and limited data are available on the mechanical response (i.e., deformation) of the epithelium during airway reopening. The goal of this study is to use image-based finite element models of cell deformation during airway reopening to investigate how cell morphology and mechanics influence the risk of cell injury/necrosis. Confocal microscopy images of EpC in subconfluent and confluent monolayers were used to generate morphologically accurate three-dimensional finite element models. Hydrodynamic stresses on the cells were calculated from boundary element solutions of bubble propagation in a fluid-filled parallel-plate flow channel. Results indicate that for equivalent cell mechanical properties and hydrodynamic load conditions, subconfluent cells develop higher membrane strains than confluent cells. Strain magnitudes were also found to decrease with increasing stiffness of the cell and membrane/cortex region but were most sensitive to changes in the cell's interior stiffness. These models may be useful in identifying pharmacological treatments that mitigate cell injury during airway reopening by altering specific biomechanical properties of the EpC.

Modulation of the pro-inflammatory phenotype of CFTR mutated epithelial cells

Background Cystic fibrosis (CF) is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). dF508 CFTR deficient targeting to the cell membrane may activate the cellular stress response and induce a pro-inflammatory phenotype by activating potent transcription factors, including NF-κB In the lung, elafin and SLPI are important anti-proteases known for their anti-microbial and anti-inflammatory (anti-NFκB) properties. The aim of this study is to assess whether correction of dF508-CFTR pro-inflammatory phenotype is possible using elafin or SLPI. Methods Human epithelial cells and CFTR over-expression: a : IB3 (dF508-CFTR over-expressers) and S9 control bronchial epithelial cells; b: over-expression of WT-CFTR and dF508-CFTR in A549 alveolar cells and BEAS-2B bronchial cells using recombinant adenovirus (Ad) vectors. Measurement of ‘CF cells’ pro-inflammatory phenotype: Sub-confluent epithelial cells were either mock-infected or infected with Ad-NF-κB-luciferase. 24 hrs later, cells were either mock-stimulated or stimulated with increased concentrations of TNF-α. Secretion of IL-8 and/or luciferase activity were then measured. Modulation of CF phenotype with antiproteases: Whenever possible, the modulation of the pro-inflammatory phenotype of ‘CF cells’ was attempted by using Ad derived elafin or SLPI. Cells were infected at subconfluence with 50 MOI of Ad. After 24 hrs, cells were either further mock-infected or infected with Ad-NF-kB-luciferase and/or stimulated with TNF-α. As above, secretion of IL-8 and/or luciferase activity were measured. Results IB3 dF508-CFTR cells secreted 4- to 6-fold more IL-8 than S9 corrected cells when stimulated with 1, 10 or 50ng/ml of TNF-α. BEAS-2B- or A549-Ad-dF508-CFTR cells also secreted high levels of IL-8, when stimulated with low levels of TNF-α, when compared to BEAS-2B- or A549-Ad-WT-CFTR controls, respectively. The IL-8 differential output in ‘CF cells’ seems mediated by the NF-kB pathway in mutated IB3 cells (2- to 4-fold increase in luciferase activity, when compared to S9-corrected cells). Conclusion We confirm that the dF508-CFTR mutation confers a pro-inflammatory phenotype to bronchial and alveolar epithelial cells which may be modulated by anti-proteases.

Mesenchymal stem cell interaction with a non‐woven hyaluronan‐based scaffold suitable for tissue repair

The fabrication of biodegradable 3-D scaffolds enriched with multipotent stem cells seems to be a promising strategy for the repair of irreversibly injured tissues. The fine mechanisms of the interaction of rat mesenchymal stem cells (rMSCs) with a hyaluronan-based scaffold, i.e. HYAFF(R)11, were investigated to evaluate the potential clinical application of this kind of engineered construct. rMSCs were seeded (2 x 10(6) cells cm(-2)) on the scaffold, cultured up to 21 days and analysed using appropriate techniques. Light (LM), scanning (SEM) and transmission (TEM) electron microscopy of untreated scaffold samples showed that scaffolds have a highly porous structure and are composed of 15-microm-thick microfibres having a rough surface. As detected by trypan blue stain, cell adhesion was high at day 1. rMSCs were viable up to 14 days as shown by CFDA assay and proliferated steadily on the scaffold as revealed by MTT assay. LM showed rMSCs in the innermost portions of the scaffold at day 3. SEM revealed a subconfluent cell monolayer covering 40 +/- 10% of the scaffold surface at day 21. TEM of early culture showed rMSCs wrapping individual fibres with regularly spaced focal contacts, whereas confocal microscopy showed polarized expression of CD44 hyaluronan receptor; TEM of 14-day cultures evidenced fibronexus formation. Immunohistochemistry of 21-day cultures showed that fibronectin was the main matrix protein secreted in the extracellular space; decorin and versican were seen in the cell cytoplasm only and type IV collagen was minimally expressed. The expression of CD90, a marker of mesenchymal stemness, was found unaffected at the end of cell culture. Our results show that HYAFF(R)11 scaffolds support the adhesion, migration and proliferation of rMSCs, as well as the synthesis and delivery of extracellular matrix components under static culture conditions without any chemical induction. The high retention rate and viability of the seeded cells as well as their fine modality of interaction with the substrate suggest that such scaffolds could be potentially useful when wide tissue defects are to be repaired as in the case of cartilage repair, wound healing and large vessel replacement.

Gene expression signatures of a fibroblastoid preosteoblast and cuboidal osteoblast cell model compared to the MLO-Y4 osteocyte cell model

In the osteoblast 2T3 cell model, 326 genes significantly increase in expression as subconfluent fibroblastic 2T3 cells become confluent and cuboidal. This gene set includes BMP2/4, Dlx2/5, Runx2, Osterix and Lrp5, as well as TGFβ regulated genes. Both activated or total nuclear Smad158 and Smad2 levels increase as they become confluent, and β-catenin protein expression increases as 2T3 cells become confluent, reflecting a set of genes involved in early preosteoblast to osteoblast commitment, as observed in vitro and in vivo. Gene Set Enrichment Analysis (GSEA) demonstrated that this 326 dataset is very similar to several early osteoblast geneset signatures. The MLO-Y4 cell model is a well-known in vitro osteocyte model. The MLO-Y4 expression pattern was directly compared with the 2T3 osteoblast cell model. 181 genes that are highly expressed in MLO-Y4 osteocytes compared to osteoblasts were identified. Very few genes expressed in MLO-Y4 cells are found in osteocytes directly isolate from bone, suggesting that osteocyte specific gene programs most likely require the osteocytes to be embedded in the proper mineralized matrix. The MLO-Y4 dataset includes few established in vivo osteocyte markers, but does include several transcription factors such as Vitamin D receptor, Tcf7, and Irx5, whose expression was confirmed in osteocytes in vivo. Gene expression signatures in MLO-Y4 cells, as determined by functional clustering and interaction maps, suggest active prostaglandin-PKA pathways, genes involved in dendrite formation, acute/defense response pathways, TGFβ signaling, and interferon/chemokine pathways. GSEA demonstrated that MLO-Y4 expression pattern is similar to macrophages, mesenchymal fibroblasts, and early osteoblasts.

Two Characteristic Regimes in Frequency-Dependent Dynamic Reorientation of Fibroblasts on Cyclically Stretched Substrates

Cells adherent on a cyclically stretched substrate with a periodically varying uniaxial strain are known to dynamically reorient nearly perpendicular to the strain direction. We investigate the dynamic reorientation of rat embryonic and human fibroblast cells over a range of stretching frequency from 0.0001 to 20 s −1 and strain amplitude from 1% to 15%. We report quantitative measurements that show that the mean cell orientation changes exponentially with a frequency-dependent characteristic time from 1 to 5 h. At subconfluent cell densities, this characteristic time for reorientation shows two characteristic regimes as a function of frequency. For frequencies below 1 s −1, the characteristic time decreases with a power law as the frequency increases. For frequencies above 1 s −1, it saturates at a constant value. In addition, a minimum threshold frequency is found below that no significant cell reorientation occurs. Our results are consistent for the two different fibroblast types and indicate a saturation of molecular mechanisms of mechanotransduction or response machinery for subconfluent cells within the frequency regime under investigation. For confluent cell layers, we observe similar behaviors of reorientation under cyclic stretch but no saturation in the characteristic time with frequency, suggesting that cell-cell contacts can play an important role in the response machinery of cells under mechanical strain.

α2β1 Integrin Regulates Lineage Commitment in Multipotent Human Colorectal Cancer Cells*

The human colorectal epithelium is maintained by multipotent stem cells that give rise to absorptive, mucous, and endocrine lineages. Recent evidence suggests that human colorectal cancers are likewise maintained by a minority population of so-called cancer stem cells. We have previously established a human colorectal cancer cell line with multipotent characteristics (HRA-19) and developed a serum-free medium that induces endocrine, mucous and absorptive lineage commitment by HRA-19 cells in vitro. In this study, we investigate the role of the β1 integrin family of cell surface extracellular matrix receptors in multilineage differentiation by these multipotent human colorectal cancer cells. We show that endocrine and mucous lineage commitment is blocked in the presence of function-blocking antibodies to β1 integrin. Function-blocking antibodies to α2 integrin also blocked both HRA-19 endocrine lineage commitment and enterocytic differentiation by Caco-2 human colon cancer cells; both effects being abrogated by the MEK inhibitor, PD98059, suggesting a role for ERK signaling in α2-mediated regulation of colorectal cancer cell differentiation. To further explore the role of α2 integrin in multilineage differentiation, we established multipotent cells expressing high levels of wild-type α2 integrin or a non-signaling chimeric α2 integrin. Overexpression of wild-type α2 integrin in HRA-19 cells significantly enhanced endocrine and mucous lineage commitment, while cells expressing the non-signaling chimeric α2 integrin had negligible ability for either endocrine or mucous lineage commitment. This study indicates that the collagen receptor α2β1 integrin is a regulator of cell fate in human multipotent colorectal cancer cells.

Forchlorfenuron Alters Mammalian Septin Assembly, Organization, and Dynamics

Septins are filamentous GTPases that associate with cell membranes and the cytoskeleton and play essential roles in cell division and cellular morphogenesis. Septins are implicated in many human diseases including cancer and neuropathies. Small molecules that reversibly perturb septin organization and function would be valuable tools for dissecting septin functions and could be used for therapeutic treatment of septin-related diseases. Forchlorfenuron (FCF) is a plant cytokinin previously shown to disrupt septin localization in budding yeast. However, it is unknown whether FCF directly targets septins and whether it affects septin organization and functions in mammalian cells. Here, we show that FCF alters septin assembly in vitro without affecting either actin or tubulin polymerization. In live mammalian cells, FCF dampens septin dynamics and induces the assembly of abnormally large septin structures. FCF has a low level of cytotoxicity, and these effects are reversed upon FCF washout. Significantly, FCF treatment induces mitotic and cell migration defects that phenocopy the effects of septin depletion by small interfering RNA. We conclude that FCF is a promising tool to study mammalian septin organization and functions.

Role of Jade-1 in the Histone Acetyltransferase (HAT) HBO1 Complex

Regulation of global chromatin acetylation is important for chromatin remodeling. A small family of Jade proteins includes Jade-1L, Jade-2, and Jade-3, each bearing two mid-molecule tandem plant homology domain (PHD) zinc fingers. We previously demonstrated that the short isoform of Jade-1L protein, Jade-1, is associated with endogenous histone acetyltransferase (HAT) activity. It has been found that Jade-1L/2/3 proteins co-purify with a novel HAT complex, consisting of HBO1, ING4/5, and Eaf6. We investigated a role for Jade-1/1L in the HBO1 complex. When overexpressed individually, neither Jade-1/1L nor HBO1 affected histone acetylation. However, co-expression of Jade-1/1L and HBO1 increased acetylation of the bulk of endogenous histone H4 in epithelial cells in a synergistic manner, suggesting that Jade1/1L positively regulates HBO1 HAT activity. Conversely, small interfering RNA-mediated depletion of endogenous Jade resulted in reduced levels of H4 acetylation. Moreover, HBO1-mediated H4 acetylation activity was enhanced severalfold by the presence of Jade-1/1L in vitro. The removal of PHD fingers affected neither binding nor mutual Jade-1-HBO1 stabilization but completely abrogated the synergistic Jade-1/1L- and HBO1-mediated histone H4 acetylation in live cells and in vitro with reconstituted oligonucleosome substrates. Therefore, PHDs are necessary for Jade-1/1L-induced acetylation of nucleosomal histones by HBO1. In contrast to Jade-1/1L, the PHD zinc finger protein ING4/5 failed to synergize with HBO1 to promote histone acetylation. The physical interaction of ING4/5 with HBO1 occurred in the presence of Jade-1L or Jade-3 but not with the Jade-1 short isoform. In summary, this study demonstrates that Jade-1/1L are crucial co-factors for HBO1-mediated histone H4 acetylation.

Substance P influenced gelatinolytic activity via reactive oxygen species in human pulp cells

To investigate the effects of substance P (SP) on gelatinolytic activity of matrix metalloproteinases (MMPs) in human pulp cells. Human dental pulp cells were isolated and cultured. Subconfluent cells, between the third and sixth passages, were maintained under serum deprivation for 18 h followed by the treatment of varying doses of SP (1 pmol L(-1), 100 pmol L(-1), 10 nmol L(-1), 1 micromol L(-1) and 100 micromol L(-1)). Conditioned media were then subjected to gelatin zymography using 8% sodium dodecyl sulphate polyacrylamide gel electrophoresis minigels containing 1.5 g L(-1) gelatin. The effect of SP on intracellular reactive oxygen species (ROS) was also examined by confocal microscopy. ROS scavenger N-Acetyl-L-cysteine (NAC, 5 mmol L(-1)) was utilized to evaluate the roles of ROS pathway in mediating the impact of SP on cellular gelatinolytic activity. Data were analysed using analysis of variance with Bonferroni correction for multiple comparisons or an unpaired Student's t-test. Substance P, at levels above 1 micromol L(-1), remarkably enhanced MMP-2 activity reflected by the band migrating at 66 kDa (P < 0.05). A gelatinolytic band at approximately 44 kDa appeared to be intensified in a SP dose-dependent manner. In addition, it was demonstrated that SP could induce ROS production in pulp cells and ROS scavenger NAC was further found to significantly reduce MMP-2 activity (P < 0.05), as well as other bands of gelatinolytic proteinases. Substance P can influence gelatinolytic activity in human pulp cells via ROS pathway.

15-Deoxy-Δ12,14-prostaglandin J2 Impairs Phosphatidylcholine Synthesis and Induces Nuclear Accumulation of Thiol-modified Cytidylyltransferase

Synthesis of phosphatidylcholine, the major phospholipid of animal cell membranes, requires the key enzyme cytidylyltransferase (CCTalpha). Cysteine sulfhydryls within CCTalpha are needed for full catalytic activity. Here we show that prostaglandin 15-deoxy-Delta12,14-PGJ2 (15d-PGJ2) inactivates CCTalpha by inducing generation of reactive oxidant species and the appearance of a cross-linked CCTalpha dimer in cells. N-Acetyl-l-cysteine reduced oxidative stress, prevented CCTalpha cross-linking, and restored CCT function in 15d-PGJ2-treated cells. 15d-PGJ2 modified critical cysteine residues within CCTalpha as determined by mutagenesis studies and by incorporation of biotin-15d-PGJ2 into CCTalpha. These effects of 15d-PGJ2 were associated with CCTalpha accumulation within the nucleus. The data indicate that bioactive prostanoids significantly impair membrane phospholipid production by promoting cysteine cross-bridging within CCTalpha.

IQGAP1 Integrates Ca2+/Calmodulin and B-Raf Signaling

Ca(2+) and calmodulin modulate numerous cellular functions, ranging from muscle contraction to the cell cycle. Accumulating evidence indicates that Ca(2+) and calmodulin regulate the MAPK signaling pathway at multiple positions in the cascade, but the molecular mechanism underlying these observations is poorly defined. We previously documented that IQGAP1 is a scaffold in the MAPK cascade. IQGAP1 binds to and regulates the activities of ERK, MEK, and B-Raf. Here we demonstrate that IQGAP1 integrates Ca(2+) and calmodulin with B-Raf signaling. In vitro analysis reveals that Ca(2+) promotes the direct binding of IQGAP1 to B-Raf. This interaction is inhibited by calmodulin in a Ca(2+)-regulated manner. Epidermal growth factor (EGF) is unable to stimulate B-Raf activity in fibroblasts treated with the Ca(2+) ionophore A23187. In contrast, chelation of intracellular free Ca(2+) concentrations ([Ca(2+)](i)) significantly enhances EGF-stimulated B-Raf activity, an effect that is dependent on IQGAP1. Incubation of cells with EGF augments the association of B-Raf with IQGAP1. Moreover, Ca(2+) regulates the association of B-Raf with IQGAP1 in cells. Increasing [Ca(2+)](i) with Ca(2+) ionophores significantly reduces co-immunoprecipitation of B-Raf and IQGAP1, whereas chelation of Ca(2+) enhances the interaction. Consistent with these findings, increasing and decreasing [Ca(2+)](i) increase and decrease, respectively, co-immunoprecipitation of calmodulin with IQGAP1. Collectively, our data identify a previously unrecognized mechanism in which the scaffold protein IQGAP1 couples Ca(2+) and calmodulin signaling to B-Raf function.

Prevention of Lens Epithelial Cell Growth In Vitro Using Mibefradil-Containing PLGA Micro Particles~!2008-04-23~!2008-05-24~!2008-06-12~!

The prevention of the posterior capsule opacification is still unsolved. To interfere with proliferating cells the T-type calcium channel antagonist Mibefradil was immobilized in poly-lactic-co-glycolic-acid micro particles which were fixed at a capsular tension ring and tested in a human organ culture model as well as in human lens cells HLE-B3 in vitro. It is feasible to get a release significantly affecting cell viability and growth evaluated by MTT test and cell cycle analysis. In addition, Bionas® sensor chips were used for time-dependent adhesion experiments in living lens cells. Interestingly, the concentration of Mibefradil which inhibited subconfluent cells is not effective in confluent cells. This is an important feature for the protection of the intact tissue in the eye.