Postconfluent Cultures Research Articles

Sustained postconfluent culture of human mammary epithelial cells enriches for luminal and c-Kit+ subtypes

BackgroundA challenge in human mammary epithelial cell (HMEC) culture is sustaining the representation of competing luminal, myoepithelial, and progenitor lineages over time. As cells replicate in culture, myoepithelial cells come to dominate the composition of the culture with serial passaging. This drift in composition presents a challenge for studying luminal and progenitor cells, which are prospective cells of origin for most breast cancer subtypes.MethodsWe demonstrate the use of postconfluent culture on HMECs. Postconfluent culture entails culturing HMECs for 2–5 weeks without passaging but maintaining frequent feedings in low-stress M87A culture medium. In contrast, standard HMEC culture entails enzymatic subculturing every 3–5 days to maintain subconfluent density.ResultsWhen compared to standard HMEC culture, postconfluent culture yields increased proportions of luminal cells and c-Kit+ progenitor cells. Postconfluent cultures develop a distinct multilayered morphology with individual cells showing decreased physical deformability as compared to cells in standard culture. Gene expression analysis of postconfluent cells shows increased expression of lineage-specific markers and extracellular matrix components.ConclusionsPostconfluent culture is a novel, useful strategy for altering the lineage composition of HMECs, by increasing the proportional representation of luminal and progenitor cells. We speculate that postconfluent culture creates a microenvironment with cellular composition closer to the physiological state and eases the isolation of scarce cell subtypes. As such, postconfluent culture is a valuable tool for researchers using HMECs for breast cancer research.

111 Single cell RNA seq revealed altered cytoskeletal dynamics and the formation of cytoplasmic RNA-Lamin A bodies during keratinocyte terminal differentiation

Keratinocyte cornification and epidermal barrier formation are tightly controlled processes, requiring degradation of organelles, including the nucleus. However, the underlying molecular mechanisms are not well defined. Post-confluent cultures of rat epidermal keratinocytes (REKs) undergo spontaneous and complete differentiation, allowing visualisation and perturbation of the differentiation process in vitro. Using this model, we have shown that the actomyosin network and the non-muscle Myosin IIa - Myh9 are required for key processes involved in nuclear degradation, comprising Akt1-mediated phosphorylation of Lamin A and the dispersal of phosphorylated Lamin A into the cytoplasmic bodies followed by rapid nuclear shrinkage. Based on these finding, we hypothesised that specific changes in actomyosin dynamics and the actin cytoskeleton are required for Lamin dispersal and nuclear shrinkage during epidermal terminal differentiation. We have designed a single-cell transcriptomics pipeline to better analyse these ’sub-optimal’, dying/cornifying cells, and high-throughput confocal imaging to analyse the actin cytoskeleton and related markers during nuclear degradation. We identified changes in expression of actin remodelling genes that coincided with nuclear degradation processes and altered distribution of the actin cytoskeleton in terminally differentiating keratinocytes. We also identified widespread up-regulation of ribosomal protein mRNAs, that correlated with the formation cytoplasmic RNA bodies associating with phosphorylated lamin A. Therefore, actin dynamics, increased translation, and increased cytoplasmic RNA are all potentially involved, and may serve as signposts for key stages in nuclear degradation and end-stage terminal differentiation in keratinocytes.

Akt1-associated actomyosin remodelling is required for nuclear lamina dispersal and nuclear shrinkage in epidermal terminal differentiation

Keratinocyte cornification and epidermal barrier formation are tightly controlled processes, which require complete degradation of intracellular organelles, including removal of keratinocyte nuclei. Keratinocyte nuclear destruction requires Akt1-dependent phosphorylation and degradation of the nuclear lamina protein, Lamin A/C, essential for nuclear integrity. However, the molecular mechanisms that result in complete nuclear removal and their regulation are not well defined. Post-confluent cultures of rat epidermal keratinocytes (REKs) undergo spontaneous and complete differentiation, allowing visualisation and perturbation of the differentiation process in vitro. We demonstrate that there is dispersal of phosphorylated Lamin A/C to structures throughout the cytoplasm in differentiating keratinocytes. We show that the dispersal of phosphorylated Lamin A/C is Akt1-dependent and these structures are specific for the removal of Lamin A/C from the nuclear lamina; nuclear contents and Lamin B were not present in these structures. Immunoprecipitation identified a group of functionally related Akt1 target proteins involved in Lamin A/C dispersal, including actin, which forms cytoskeletal microfilaments, Arp3, required for actin filament nucleation, and Myh9, a component of myosin IIa, a molecular motor that can translocate along actin filaments. Disruption of actin filament polymerisation, nucleation or myosin IIa activity prevented formation and dispersal of cytoplasmic Lamin A/C structures. Live imaging of keratinocytes expressing fluorescently tagged nuclear proteins showed a nuclear volume reduction step taking less than 40 min precedes final nuclear destruction. Preventing Akt1-dependent Lamin A/C phosphorylation and disrupting cytoskeletal Akt1-associated proteins prevented nuclear volume reduction. We propose keratinocyte nuclear destruction and differentiation requires myosin II activity and the actin cytoskeleton for two intermediate processes: Lamin A/C dispersal and rapid nuclear volume reduction.

Extracellular Matrix and Integrin Expression Profiles in Fuchs Endothelial Corneal Dystrophy Cells and Tissue Model

Primary corneal endothelial cell (CEC) cultures and 3D-engineered tissue models were used to study the aberrant deposition of extracellular matrix (ECM) in a vision impairing pathology known as Fuchs endothelial corneal dystrophy (FECD). CECs were isolated from excised Descemet membranes of patients with end-stage FECD. CECs isolated from healthy corneas served as controls. Microarray gene profiling was performed on postconfluent cultures of healthy and FECD cells. Protein expression analyses were conducted on tissue models that were engineered by seeding an endothelium on previously devitalized human stromal carriers. The engineered endothelia were kept in culture for 1–3 weeks to reform the endothelial monolayer. Protein expression of integrin subunits α4, α6, αv, and β1, as well as laminin, type IV collagen, fibronectin, clusterin, and transforming growth factor β-induced protein (TGFβIp) was then assessed by immunofluorescence. Microarray analysis showed nonstatistical twofold downregulation of collagen-coding genes (COL4A4, COL8A2, and COL21A1) and a twofold upregulation of the COL6A1, laminin α3 gene LAMA3, and integrin subunit α10 gene ITGA10 in FECD cells. Fibronectin type III domain containing 4 (FNDC4) and integrin β5 (ITGB5) genes was significantly upregulated in FECD cells. Immunostainings demonstrated that the protein expression of the integrin subunits α4, α6, αv, and β1, type IV collagen, as well as laminin remained similar between native and engineered endothelia. TGFβIp expression was found on the stromal side of both FECD and healthy Descemet's membrane, and only one out of three FECD specimens was positive for the clusterin protein. Interestingly, the ECM protein fibronectin was also found to have a stronger presence on engineered FECD tissues, a result consistent with the native FECD specimens. To conclude, this study allowed to identify fibronectin deposition as one of the first steps in the pathogenesis of FECD, as defined by our engineered tissue model. This opens the way to an entirely new perspective for in vitro pharmacological testing of new therapies for FECD, the leading indication for corneal transplantation in North America.

Calcium Sensing Receptor Function Supports Osteoblast Survival and Acts as a Co‐Factor in PTH Anabolic Actions in Bone

Anabolic actions of PTH in bone involve increased deposition of mineralizing matrix. Regulatory feedback of the process may be important to maintain calcium homeostasis and, in turn, calcium may inform the process. This investigation clarified the role of calcium availability and the calcium sensing receptor (CaSR) in the anabolic actions of PTH. CaSR function promoted osteoblastic cell numbers, with lower cell numbers in post-confluent cultures of primary calvarial cells from Col1-CaSR knock-out (KO) mice, and for calvarial cells from wild-type (WT) mice treated with a calcilytic. Increased apoptosis of calvarial cells with calcilytic treatment suggested CaSR is critical for protection against stage-dependent cell death. Whole and cortical, but not trabecular, bone parameters were significantly lower in Col1-CaSR KO mice versus WT littermates. Intact Col1-CaSR KO mice had lower serum P1NP levels relative to WT. PTH treatment displayed anabolic actions in WT and, to a lesser degree, KO mice, and rescued the lower P1NP levels in KO mice. Furthermore, PTH effects on whole tibiae were inhibited by osteoblast-specific CaSR ablation. Vertebral body implants (vossicles) from untreated Col1-CaSR KO and WT mice had similar bone volumes after 4 weeks of implantation in athymic mice. These findings suggest that trabecular bone formation can occur independently of the CaSR, and that the CaSR plays a collaborative role in the PTH anabolic effects on bone. J. Cell. Biochem. 117: 1556-1567, 2016. © 2015 Wiley Periodicals, Inc.

Selective upregulation of the expression of plasma membrane calcium ATPase isoforms upon differentiation and 1,25(OH)2D3-vitamin treatment of colon cancer cells

We have previously presented co-expression of the plasma membrane calcium ATPase isoforms 4b (PMCA4b) and 1b (PMCA1b) in colon carcinoma cells, and selective upregulation of PMCA4b during differentiation initiated by short chain fatty acids or post-confluent growth. Here we show that the induction of PMCA4b expression is a characteristic feature of the post-confluency-induced differentiation of both enterocyte-type and goblet cell-type colon cancer cells. Vitamin D3 (1,25(OH)2D3) is a well-known regulator of intestinal Ca2+ absorption and of basic cell functions such as growth and differentiation in various cell types. As PMCA proteins are involved both in intestinal Ca2+ absorption and adenocarcinoma cell differentiation, we investigated the effect of 1,25(OH)2D3 on PMCA expression in enterocyte-like colon carcinoma cells, and monitored its effect on the expression of various differentiation markers. 1,25(OH)2D3 stimulated PMCA1b, but not PMCA4b expression without modulating the expression of the majority of the differentiation markers examined. Caco-2 cells differentiated in post-confluent cultures present normal enterocyte-like intestinal epithelial phenotype. To better understand the role of PMCA proteins in vectorial Ca2+ transport by enterocytes, we also studied their subcellular localization in mature polarized Caco-2 cells. Both PMCA isoforms were located to the basolateral membrane, and the PMCA-specific immunofluorescent signal was significantly higher in vitamin D3-treated cells, underlining the 1,25(OH)2D3-induced upregulation of PMCA (presumably 1b isoform) expression in differentiated Caco-2 cells. We suggest that while PMCA1b has a housekeeping function in colon cancer cells, PMCA4b participates in the reorganization of the Ca2+ signalling machinery during cell differentiation. The subcellular localization of PMCA1b and its selective 1,25(OH)2D3-dependent upregulation indicate that this isoform may have a specific role in 1,25(OH)2D3-stimulated intestinal Ca2+ absorption.

Influence of Cell Confluency on the Expression of the α4 Integrin Subunit of Retinal Pigment Epithelial Cells

Integrins are a family of transmembrane glycoproteins that mediate cell-cell and cell-extracellular matrix interactions. The integrin α4 subunit is widely expressed by cells from the immune system and its expression by non-hematopoietic cells is scarce. In the present study, gene and protein expression of this integrin subunit was characterized in proliferating and quiescent human RPE cells. Immunofluorescent studies confirm that the α4 subunit is expressed in vitro by RPE cells, a result that has been validated by immunofluorescence and FACS analyses. The accumulation of the α4 integrin at cell-cell junctions in post-confluent RPE cell cultures negatively correlated with the level of expression of the mRNA transcript. Accordingly, transient transfection analyses reveal that the α4 promoter activity is considerably reduced when RPE cells form a confluent monolayer. Moreover, transfection of recombinant constructs bearing 5’-deletions of the α4 promoter segment allows the localization of strong negative regulatory elements on the -76 to -300 region of the α4 gene suggesting that its expression is intimately linked to the proliferative state of primary cultured RPE cells.

Preparation of Pre-Confluent Retinal Cells Increases Graft Viability In Vitro and In Vivo: A Mouse Model

PurposeGraft failure remains an obstacle to experimental subretinal cell transplantation. A key step is preparing a viable graft, as high levels of necrosis and apoptosis increase the risk of graft failure. Retinal grafts are commonly harvested from cell cultures. We termed the graft preparation procedure “transplant conditions” (TC). We hypothesized that culture conditions influenced graft viability, and investigated whether viability decreased following TC using a mouse retinal pigment epithelial (RPE) cell line, DH01.MethodsCell viability was assessed by trypan blue exclusion. Levels of apoptosis and necrosis in vitro were determined by flow cytometry for annexin V and propidium iodide and Western blot analysis for the pro- and cleaved forms of caspases 3 and 7. Graft viability in vivo was established by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and cleaved caspase 3 immunolabeling of subretinal allografts.ResultsPre-confluent cultures had significantly less nonviable cells than post-confluent cultures (6.6%±0.8% vs. 13.1%±0.9%, p<0.01). Cell viability in either group was not altered significantly following TC. Caspases 3 and 7 were not altered by levels of confluence or following TC. Pre-confluent cultures had low levels of apoptosis/necrosis (5.6%±1.1%) that did not increase following TC (4.8%±0.5%). However, culturing beyond confluence led to progressively increasing levels of apoptosis and necrosis (up to 16.5%±0.9%). Allografts prepared from post-confluent cultures had significantly more TUNEL-positive cells 3 hours post-operatively than grafts of pre-confluent cells (12.7%±3.1% vs. 4.5%±1.4%, p<0.001). Subretinal grafts of post-confluent cells also had significantly higher rates of cleaved caspase 3 than pre-confluent grafts (20.2%±4.3% vs. 7.8%±1.8%, p<0.001).ConclusionPre-confluent cells should be used to maximize graft cell viability.

EGFR and IL-1 Signaling Synergistically Promote Keratinocyte Antimicrobial Defenses in a Differentiation-Dependent Manner

Ligands of the EGF family regulate autocrine keratinocyte proliferation, and IL-1 family cytokines orchestrate epithelial defense responses. Although members of both families are overexpressed in wound healing and psoriasis, their roles in regulating the innate immune functions of keratinocytes remain incompletely explored. Using sensitive assays, we found significant increases of heparin-binding EGF-like growth factor, transforming growth factor-α, and amphiregulin mRNA and protein in lesional psoriasis compared with uninvolved or control skin. In normal human keratinocyte (NHK) monolayers, EGFR ligands were ineffective in inducing DEFB4, S100A7, and CCL20 mRNAs and human β-defensin (hBD)-2 peptide. Combined with IL-1α, however, EGFR ligands provoked 250 × more DEFB4 and CCL20 and a 9-fold rise in S100A7 mRNA relative to the EGFR ligand alone. This synergy was also reflected in secreted hBD-2 protein, both from NHK and reconstituted human epidermis. Keratinocyte differentiation was critical for these responses, as postconfluent NHK yielded mRNA and protein levels an order of magnitude greater than subconfluent cells. Differentiation also influenced signal transduction, with subconfluent cells using NF-κB and postconfluent cells using EGFR, MEK1/2, and p38. We propose that EGFR ligands are important modifiers of IL-1 activity, synergizing with IL-1 to stimulate epidermal production of hBD-2, S100A7, and CCL20, three of the most upregulated transcripts in psoriatic plaques.

Developmentally regulated inhibition of cell cycle progression by glucocorticoids through repression of cyclin a transcription in primary osteoblast cultures

Synthetic glucocorticoids (GCs) like dexamethasone (DEX) are effective immunosuppressants indicated for autoimmune and inflammatory diseases. However, they often promote osteoporosis and bone fractures. Here, we investigated the anti-mitogenic effect of GCs in primary osteoblast cultures. DEX did not affect cell cycle progression in confluent (day 2), or early post-confluent cultures. Starting on day 5, however, DEX strongly inhibited the G1/S cell cycle transition. Day 5 also marked the beginning of a ∼2-day commitment stage, during which cultures acquired resistance to the inhibitory effect of DEX on mineralization. Considering the importance of Wnt signaling in osteoblast development, we analyzed the effect of DEX on the Wnt pathway. DEX treatment did not inhibit the TOPGAL Wnt reporter before, but only during and after the commitment stage. However, this inhibition was not associated with repression of neither cyclin D1 nor c-Myc mRNA, well-established Wnt targets regulating cell cycle progression. On the other hand, acute (24-h) and chronic (7 days) DEX treatment significantly reduced both the mRNA and protein levels of cyclin A, another cell cycle regulator. Moreover, cyclin A repression by DEX was not observed before, but only during and after the commitment stage. Using gel shift and reporter assays, we identified an ATF/CREB-binding site critical for the DEX-mediated repression of cyclin A transcription. Furthermore, and similar to cyclin A, Atf4 expression was repressed by DEX only during and after commitment. Our data suggest that GCs attenuate cell cycle progression in osteoblasts in a developmental stage-specific manner by repressing Atf4-dependent cyclin A gene expression.

Cadmium‐induced hormetic effect in differentiated Caco‐2 cells: ERK and p38 activation without cell proliferation stimulation

Cadmium (Cd) is a toxic metal that enters the food chain. Following oral ingestion, the intestinal epithelium may in part protect against Cd toxicity but is also a target tissue. Using human enterocytic-like Caco-2 cells, we have previously shown differences in sensitivity to Cd according to the differentiation status. The present study focuses on Cd effects on differentiated cells. Concentration and time-dependent increases in MTT (3-[4,5-dimethyl-2-thiazol-2-yl]-2,5-diphenyltetrazolium bromide assay) activity were observed in post-confluent cultures exclusively, with a twofold maximal stimulation in 21-day-old cells exposed to 10 microM Cd for 24 h. No concomitant increase in [methyl-(3)H] thymidine incorporation was noted and Cd did not modify cell distribution in the cell-cycle phases. However, Cd-induced increase in MTT activity was inhibited by cycloheximine as well as by inhibitors of ERK1/2 and p38, but not by that of JNK. Consistently, Cd increased the levels of ERK1/2 and p38 phosphorylation. Inhibition of Ras-GTP or PI3K enhanced the stimulatory effect of Cd, whereas mTOR inhibition had no effect. Inhibition of G protein-phospholipase and PKC decreased MTT stimulation. These results show a hormesis-like stimulation of Cd on MTT activity in differentiated intestinal cells exclusively. This effect is not related to cell proliferation but more likely to increased protein synthesis which involves ERK1/2 and p38 cascades and possibly PLC-beta signaling pathways. Because growth-related differentiation of intestinal cells is linked to the selective and sequential activation of MAPKs, the impacts that these Cd-induced perturbations in signaling pathways may have on intestinal functions clearly deserve to be investigated.

Mutations in LTBP4 Cause a Syndrome of Impaired Pulmonary, Gastrointestinal, Genitourinary, Musculoskeletal, and Dermal Development

We report recessive mutations in the gene for the latent transforming growth factor-beta binding protein 4 (LTBP4) in four unrelated patients with a human syndrome disrupting pulmonary, gastrointestinal, urinary, musculoskeletal, craniofacial, and dermal development. All patients had severe respiratory distress, with cystic and atelectatic changes in the lungs complicated by tracheomalacia and diaphragmatic hernia. Three of the four patients died of respiratory failure. Cardiovascular lesions were mild, limited to pulmonary artery stenosis and patent foramen ovale. Gastrointestinal malformations included diverticulosis, enlargement, tortuosity, and stenosis at various levels of the intestinal tract. The urinary tract was affected by diverticulosis and hydronephrosis. Joint laxity and low muscle tone contributed to musculoskeletal problems compounded by postnatal growth delay. Craniofacial features included microretrognathia, flat midface, receding forehead, and wide fontanelles. All patients had cutis laxa. Four of the five identified LTBP4 mutations led to premature termination of translation and destabilization of the LTBP4 mRNA. Impaired synthesis and lack of deposition of LTBP4 into the extracellular matrix (ECM) caused increased transforming growth factor-beta (TGF-beta) activity in cultured fibroblasts and defective elastic fiber assembly in all tissues affected by the disease. These molecular defects were associated with blocked alveolarization and airway collapse in the lung. Our results show that coupling of TGF-beta signaling and ECM assembly is essential for proper development and is achieved in multiple human organ systems by multifunctional proteins such as LTBP4.

The first extracellular domain of the tumour stem cell marker CD133 contains an antigenic ganglioside-binding motif

Prominin 1/CD133 is a marker of transplantable cancer stem cells. We have generated anti-peptide antibodies against a N-terminal epitope of CD133 belonging to a ganglioside-binding domain. The labelling of colon cancer cells with these antibodies was inhibited by various gangliosides including GM1 and GD3, but not GT1b. CD133 immunolabelling progressively decreased to undetectable levels in post-confluent cultures, possibly through ganglioside-mediated epitope masking since the staining was partially recovered after chemical disruption of lipid rafts. We suggest that selected gangliosides could modulate the accessibility of CD133 and regulate cell–cell contacts involving CD133+ stem cells at the earliest steps of tumour development.

The role of protein tyrosine phosphorylation in the cell–cell interactions, junctional permeability and cell cycle control in post-confluent bovine corneal endothelial cells

Cell–cell interaction, junctional permeability and contact inhibition are important mechanisms that allow corneal endothelial cells to maintain stable corneal hydration status and also keep these cells in non-proliferative status. Protein tyrosine phosphatases (PTPs) are well known to play an important role in regulating cell–cell contacts, growth and differentiation. Inhibition of PTPs activity by a general PTP inhibitor has been proved to trigger post-confluent rat corneal endothelial cells to reenter cell cycles. In this study, we aimed to evaluate whether protein tyrosine phosphorylation is involved in cell–cell interactions, junctional permeability and cell cycle control in post-confluent, contact inhibited bovine corneal endothelial cells. Confluent cultures of bovine corneal endothelial cells were treated with different concentrations of general phosphatase inhibitor, sodium orthovanadate (vanadate) for several different durations. Protein tyrosine phosphorylation was confirmed by Western blot analysis. Immunocytochemistry was used to evaluate the effect of vanadate on adherens-type junctional proteins by staining of p120, N-cadherin and α-catenin. Paracelluar permeability was evaluated by transepithelial electric resistance. The effect of vanadate on cell cycle progression was confirmed by immunostaining of Ki67. Western blot analysis was used to evaluate the expression level of cell-cycle-associated proteins, including PCNA, cyclin D1, cyclin E and cyclin A. Time-dependent effects of vanadate on protein tyrosine phosphorylation were confirmed by Western blot analysis. ICC demonstrated the effect of vanadate on the disruption of p120, N-cadherin and α-catenin. Time- and dose-effects of vanadate on the severity of p120 disruption were also found. TER demonstrated the time- and dose-effect of vanadte on paracellular permeability. Although cell–cell junctions can be broken through by vanadate, no significant increase of Ki67 positive cells was found among the control group and all groups with different concentrations/durations of vanadate treatment. Western blot also showed no change of PCNA, cyclin D1, cyclin E and cyclin A after treatment with vanadate. In conclusion, protein tyrosine phosphatase inhibition can induce time-dependent release of cell–cell contacts and increase transepithelial permeability in post-confluent cultures of bovine corneal endothelial cells. However, such phenomenon is not enough to promoted cell cycle progression as seen in rat corneal endothelial cells.

In vitro differentiation of HT‐29 M6 mucus‐secreting colon cancer cells involves a trychostatin A and p27KIP1‐inducible transcriptional program of gene expression

Tumor cell dedifferentiation-such as the loss of cell-to-cell adhesion in epithelial tumors-is associated with tumor progression. To better understand the mechanisms that maintain carcinoma cells in a differentiated state, we have dissected in vitro differentiation pathways in the mucus-secretor HT-29 M6 colon cancer cell line, which spontaneously differentiates in postconfluent cultures. By lowering the extracellular calcium concentration to levels that prevent intercellular adhesion and epithelial polarization, our results reveal that differentiation is calcium-dependent and involves: (i) a process of cell cycle exit to G(0) and (ii) the induction of a transcriptional program of differentiation gene expression (i.e., mucins MUC1 and MUC5AC, and the apical membrane peptidase DPPIV). In calcium-deprived, non-differentiated postconfluent cultures, differentiation gene promoters are repressed by a trichostatin A (TSA)-sensitive mechanism, indicating that loss of gene expression by dedifferentiation is driven by histone deacetylases (HDAC). Since TSA treatment or extracellular calcium restoration allow gene promoter activation to similar levels, we suggest that induction of differentiation is one mechanism of HDAC inhibitor antitumor action. Moreover, transcriptional de-repression can also be induced in non-differentiating culture conditions by overexpressing the cyclin-dependent kinase inhibitor p27(KIP1), which is normally induced during spontaneous differentiation. Since p27(KIP1) downregulation in colon cancer is associated with poor prognosis independently of tumor cell division rates, we propose that p27 (KIP1) may prevent tumor progression by, at least in part, enhancing the expression of some differentiation genes. Therefore, the HT-29 M6 model allows the identification of some basic mechanisms of cancer cell differentiation control, so far revealing HDAC and p27(KIP1) as key regulatory factors of differentiation gene expression.

Regulation of cGMP-dependent Protein Kinase Expression by Rho and Krüppel-like Transcription Factor-4

Type I cGMP-dependent protein kinase (PKG I) plays a major role in vascular homeostasis by mediating smooth muscle relaxation in response to nitric oxide, but little is known about the regulation of PKG I expression in smooth muscle cells. We found opposing effects of RhoA and Rac1 on cellular PKG I expression: (i) cell density-dependent changes in PKG I expression varied directly with Rac1 activity and inversely with RhoA activity; (ii) RhoA activation by calpeptin suppressed PKG I, whereas RhoA down-regulation by small interfering RNA increased PKG I expression; and (iii) PKG I promoter activity was suppressed in cells expressing active RhoA or Rho-kinase but was enhanced in cells expressing active Rac1 or a dominant negative RhoA. Sp1 consensus sequences in the PKG I promoter were required for Rho regulation and bound nuclear proteins in a cell density-dependent manner, including the Krüppel-like factor 4 (KLF4). KLF4 was identified as a major trans-acting factor at two proximal Sp1 sites; active RhoA suppressed KLF4 DNA binding and trans-activation potential on the PKG I promoter. Experiments with actin-binding agents suggested that RhoA could regulate KLF4 via its ability to induce actin polymerization. Regulation of PKG I expression by RhoA may explain decreased PKG I levels in vascular smooth muscle cells found in some models of hypertension and vascular injury.

Expression of lysosome-associated membrane protein 1 (Lamp-1) and galectins in human keratinocytes is regulated by differentiation

Lysosomes and their components are suspected to be involved in epidermal differentiation. In this study, lysosomal enzyme activities, expression of the lysosome-associated membrane protein 1 (Lamp-1) and expression of the epidermal galectins-1, -3 and -7 were investigated in human keratinocytes cultured at different cell densities (subconfluence, confluence and postconfluence) in order to induce differentiation. Detected by Western blot and immunofluorescence, Lamp-1 expression is transiently upregulated at culture confluence, but reduced at postconfluence. Northern blot analyses performed on subconfluent, confluent and post-confluent cultures of keratinocytes show that Lamp-1 mRNA expression is also upregulated at culture confluence, but downregulated at postconfluence. Measurements of lysosomal enzyme activities indicate a transient upregulation at culture confluence, whereas cathepsins B, C and L are particularly downregulated at postconfluence. Cell density and differentiation of epidermal cells also differentially regulates galectin expression in autocrine cultures. As the expression of galectin-1 mRNA is high in subconfluent cells, it is assumed to be associated with their proliferative state. On the other hand, as the mRNA levels for galectins-3 and -7 are notably upregulated at culture confluence (galectin-7) or at postconfluence (galectin-3), their expression is thought to be related to the differentiated state of keratinocytes. However, we collected evidence by confocal microscopy that galectin-3 and Lamp-1 do not colocalize in vitro in keratinocytes. Altogether, our results suggest that the upregulated Lamp-1 expression at confluence could be involved in keratinocyte differentiation, but apparently not through interaction with galectin-3.

Cytotoxicity of sphingoid marine compound analogs in mono- and multilayered solid tumor cell cultures

A subset of four synthetic sphingoid marine compound analogs was chosen from a preliminary in vitro cytotoxicity study for further analysis. The selected analogs were initially screened in monolayer cultures for their anticancer potential against a panel of eight human tumor cell lines, ovarian, colon and lung cancer, squamous cell carcinoma and leukemia producing IC50 values ranging from 1.5 to 6.9 microM. In a secondary screening, the sphingoid analogs were evaluated against multilayered postconfluent cultures of A2780 ovarian cancer and WiDr colon cancer cells. In this model, compounds 5 and 8 were the most active derivatives showing EC50 values in the range 25-32 microM. The performance of 5 and 8 against both cell lines was not dependent on the cell culture model as shown with resistance factor values in the range 8-12. Cell cycle studies in HL60 leukemia cells showed an arrest in G(0)/G1 at a low drug concentration (3 microM) but accumulation in S phase at a high drug concentration (9 microM). It can be concluded that the analogs showed a cell line independent activity, with an apparent selectivity against cells grown in more physiological three-dimensional condition compared to standard anticancer drugs.

Kruppel-like Factor 4 Abrogates Myocardin-induced Activation of Smooth Muscle Gene Expression

Platelet-derived growth factor BB (PDGF-BB) has been shown to be an extremely potent negative regulator of smooth muscle cell (SMC) differentiation. Moreover, previous studies have demonstrated that the Kruppel-like transcription factor (KLF) 4 potently represses the expression of multiple SMC genes. However, the mechanisms whereby KLF4 suppresses SMC gene expression are not known, nor is it clear whether KLF4 contributes to PDGF-BB-induced down-regulation of SMC genes. The goals of the present studies were to determine the molecular mechanisms by which KLF4 represses expression of SMC genes and whether it contributes to PDGF-BB-induced suppression of these genes. Results demonstrated that KLF4 markedly repressed both myocardin-induced activation of SMC genes and expression of myocardin. KLF4 was rapidly up-regulated in PDGF-BB-treated, cultured SMC, and a small interfering RNA to KLF4 partially blocked PDGF-BB-induced SMC gene repression. Both PDGF-BB and KLF4 markedly reduced serum response factor binding to CArG containing regions within intact chromatin. Finally, KLF4, which is normally not expressed in differentiated SMC in vivo, was rapidly up-regulated in vivo in response to vascular injury. Taken together, results indicate that KLF4 represses SMC genes by both down-regulating myocardin expression and preventing serum response factor/myocardin from associating with SMC gene promoters, and suggest that KLF4 may be a key effector of PDGF-BB and injury-induced phenotypic switching of SMC.

Glucocorticoids Inhibit the Transcriptional Activity of LEF/TCF in Differentiating Osteoblasts in a Glycogen Synthase Kinase-3β-dependent and -independent Manner

Glucocorticoids, widely used as immune suppressors, cause osteoporosis by inhibiting bone formation. In MC3T3-E1 osteoblast-like cultures, dexamethasone (DEX) activates glycogen synthase kinase-3beta (GSK3beta) and inhibits a differentiation-related cell cycle that occurs at a commitment stage immediately after confluence. Here we show that DEX inhibition of the differentiation-related cell cycle is associated with a decrease in beta-catenin levels and inhibition of LEF/TCF-mediated transcription. These inhibitory activities are no longer observed in the presence of lithium, a GSK3beta inhibitor. DEX decreased the serum-responsive phosphorylation of protein kinase B/Akt-Ser(473) within minutes, and this inhibition was also observed after 12 h. When the phosphatidylinositol 3-kinase (PI3K)/Akt pathway was inhibited by wortmannin, DEX no longer inhibited beta-catenin levels. Furthermore, DEX-mediated inhibition of LEF/TCF transcriptional activity was attenuated in the presence of dominant negative forms of either PI3K or protein kinase B/Akt. These results suggest cross-talk between the PI3K/Akt and Wnt signaling pathways. Consistent with a role for Wnt signaling in the osteoblast differentiation-related cell cycle, wortmannin partially negated the DEX inhibition of this cell cycle. DEX also induced histone deacetylase (HDAC) 1, which is known to inhibit LEF/TCF transcriptional activity. Overexpression of HDAC1 negated the inhibitory effect of DEX on LEF/TCF transcriptional activity. In the presence of trichostatin A, a deacetylase inhibitor, DEX-mediated inhibition of the differentiation-related cell cycle was partially negated. When administered together, wortmannin and trichostatin A completely negated the inhibitory effect of DEX on the differentiation-related cell cycle. These results suggest that inhibition of a PI3K/Akt/GSK3beta/beta-catenin/LEF axis and stimulation of HDAC1 cooperate to mediate the inhibitory effect of DEX on Wnt signaling and the osteoblast differentiation-related cell cycle.