Low Calcium Medium Research Articles

The in vitro behaviour and patterns of colony formation of murine epithelial stem cells

The mechanisms of renewal of skin and mucosal epithelia in vivo are associated with hierarchies of stem and amplifying cells organized in distinct spatial patterns. Stem and amplifying characteristics persist after isolation and growth of human keratinocytes in vitro but the pattern for murine keratinocytes has been less clear. Murine keratinocytes were grown in low calcium media and examined for their patterns of colony morphologies. We consistently identified three types of colonies, one of which contains concentric zones of amplifying and differentiated cells surrounding a central zone of cells that have patterns of expression and behavioural characteristic of stem cells. This zonal organization facilitated analysis of stem cell formation and loss. Cells in the central stem cell zone undergo rapid symmetric divisions but expansion of this population is partially limited by their peripheral transition into amplifying cells. A striking feature of central zone cells is their enhanced apoptotic susceptibility and stem cell expansion limited by consistently high background rates of apoptosis. This occasionally reaches catastrophic levels with elimination of the entire central zone. In vitro amplification of stem cells for the generation of engineered tissue has tended to focus on control of asymmetric division but these findings suggest that development of mechanisms protecting stem cells from apoptotic changes are also likely to be of particular value.

Culture requirements of prostatic epithelial cell lines for acinar morphogenesis and lumen formation in vitro: Role of extracellular calcium

Three-dimensional (3D) culture of benign prostatic epithelial cell lines can recapitulate acinar morphogenesis in vitro, but the broad applicability of this approach has not been described. The present studies examine the culture conditions important for prostatic acinar morphogenesis in vitro and the role of extracellular calcium in this process. With optimized culture conditions, RWPE-1, pRNS-1-1, PZ-HPV-7, PNT1A, BPH-1, and PrEC were analyzed for their ability to undergo acinar morphogenesis in 3D culture and by immunoblotting. RWPE-1 cells were further examined for the effects of calcium on morphology, E-cadherin membrane localization and multicellular layering in 2D culture and for acinar morphogenesis, luminal apoptosis, and luminal filling in 3D. Cell lines grown in low-calcium medium have the ability to form acinar structures with lumens, which correlates with E-cadherin expression, but low calcium is not required for this process. Adding CaCl(2) to the medium strongly inhibits lumen formation, luminal apoptosis and induces luminal filling, and luminal filling is blocked by an interfering antibody. Optimized medium composition allows nearly all seeded RWPE-1 cells to undergo acinar morphogenesis, forming consistent structures representative of normal adult prostate glands. Low-calcium-containing medium appears selective for cells capable of undergoing acinar morphogenesis in vitro, and branching and luminal space within the acini are strongly influenced by extracellular calcium levels, likely through the actions of E-cadherin. These results provide important information about a relevant in vitro model with which to study prostate development and carcinogenesis and highlight the importance of extracellular calcium in regulating 3D morphology.

Isolation and characterization of human gastric cell lines with stem cell phenotypes

The aim of this study was to develop an in vitro human gastric stem and/or progenitor cell model that may be used to study the mechanism of gastric carcinogenesis induced by Helicobacter pylori infection. Human gastric biopsy was minced and digested with collagenase and dispase and cultured in a low-calcium medium (serum-free keratinocyte medium; keratinocyte-SFM) supplemented with N-acetyl-L-cysteine and L-ascorbic acid 2-phosphate. Actively proliferating epithelial colonies with sustained growth were isolated and characterized for karyotype and phenotypes related to stem cell characteristics including proliferation and differentiation potential, ability of anchorage-independent growth (AIG), gap junctional intercellular communication (GJIC) and the expression of Oct-4, a transcription factor previously shown to be expressed in embryonic stem cells, adult stem cells and undifferentiated tumor cells. To study the carcinogenic effect of H. pylori infection, gastric stem and/or progenitor cells were incubated with H. pylori culture products and/or N-methyl-N-nitro-N-nitrosoguanidine (MNNG), a chemical carcinogen, to see the telomerase activation. Multiple cell lines with stem cell features were isolated by this new cell culture method. The results based on detailed characterization of one cell clone, KMU-GI2, revealed stem cell features of these cells. The initial clone contained mostly undifferentiated epithelial-like cells, which, upon subculture and propagation, gave rise to a heterogeneous cell population. Single cell-derived subclones, similar to the parental population, retained high differentiation potential and were capable of giving rise to many morphologically different cell types (i.e. epithelial-like, glial or neuron-like, round and various peculiar-shaped cells). Although these cells were normal in karyotype and competent in GJIC, they had the ability to grow in soft agar. Cells expressing epithelial membrane antigen (EMA), mucin 5AC, glial fibrillary acidic protein (GFAP), cytokeratin-18 (CK-18), trefoil factor 1 (TFF-1) and Oct-4 were found in the cell culture, but not E-cadherin-, gastrin- or telomerase-expressing cells. Furthermore, spontaneously immortalized non-tumorigenic clones could be derived from the cell population. After treating these cell cultures with the chemical carcinogen, MNNG and H. pylori culture products for 5 days, telomerase activity and telomerase mRNA expression were significantly elevated, while treatment with either of them showed no effect. The new cell culture method can be used to develop gastric epithelial cell clones with sustained growth from endoscopic biopsy. The gastric cell clone showed several stem and/or progenitor cell phenotypes (i.e. the ability of AIG, high differentiation capacity, high susceptibility to spontaneous immortalization and the expression of Oct-4). The telomerase expression in these gastric stem and/or progenitor cells can be upregulated by exposure to H. pylori culture products and MNNG, an important step in neoplastic transformation. These results show that putative human gastric stem and/or progenitor cell clones can be developed by our method and these cells could be useful for studying the mechanisms of human gastric carcinogenesis including the mechanism of action of H. pylori, as well as the regulation of the proliferation and differentiation of human gastric mucosa.

Wnt5a secretion stimulated by the extracellular calcium-sensing receptor inhibits defective Wnt signaling in colon cancer cells

To understand the role of the colonic extracellular calcium-sensing receptor (CaSR) in calcium chemoprotection against colon cancer, we activated the CaSR with 5 mM Ca(2+) on HT-29 cells, an adenocarcinoma cell line. High Ca(2+) stimulated the upregulation (as assessed by RT-PCR) and the secretion of Wnt5a (assessed by Western blot), a noncanonical Wnt family member. Inhibiting CaSR activity with a short interfering RNA (siRNA) duplex against the CaSR reduced CaSR protein and prevented the secretion of Wnt5a. Dominant negative CaSR (R185Q) or siRNA blocked the high Ca(2+)-mediated inhibition of the beta-catenin reporter TOPflash. The CaSR/Wnt5a inhibition of beta-catenin reporter was prevented by dominant negative ubiquitin ligase seven in absentia homolog 2 (Siah2). In low-calcium medium, overexpressing Wnt5a increased Siah2 amplicons and protein. Inducing the expression of full-length adenomatous polyposis coli (APC) prevented CaSRmediated increases of Siah2 and Wnt5a. Overexpressing the receptor tyrosine kinase-like orphan receptor 2 (Ror2) increased Wnt5a and CaSR-mediated inhibition of TOPflash. Conditioned medium from Wnt5a-transfected cells added to HT-29 cells in low-Ca(2+) medium inhibited the beta-catenin reporter. This inhibition was blocked dose responsively by Frizzled-8/Fc chimeric antibody. Overexpression of Ror2 in HT-29 cells in low-Ca(2+) medium increased the inhibition of beta-catenin reporter caused by recombinant Wnt5a protein compared with addition of Wnt5a protein alone. Our findings demonstrate that APC status plays a key role as a determinant of Wnt5a secretion and suggest that CaSR-mediated secretion of Wnt5a will inhibit defective Wnt signaling in APC-truncated cells in an autocrine manner.

A Novel Monoclonal Antibody Against the Second Extracellular Loop of Occludin Disrupts Epithelial Cell Polarity

The tight junction (TJ) regulates epithelial cell polarity and paracellular permeability. In the present study, to investigate whether the second extracellular loop of occludin affects the localization of carcinoembryonic antigen (CEA) and CD26 expressed on apical membranes, and the fence function of the TJ, the human intestinal epithelial cell line T84 was treated with the monoclonal anti-occludin antibody (MAb) 1H8, corresponding to the second extracellular loop of occludin. In T84 cells treated with MAb 1H8, occludin disappeared, and CEA and CD26 were observed to diffuse from the apical membrane to the basolateral membrane. Furthermore, a decrease in the fence function of TJ was observed without changes in the TJ strands and barrier function. When T84 cells precultured in low calcium (Ca) medium were recultured in normal Ca medium in the presence of MAb 1H8, recruitment of occludin to the apical-most membranes and recovery in distribution of CEA and CD26 were markedly retarded compared with the control. These results suggested that MAb 1H8 against the second extracellular loop of occludin selectively affected formation of the apical/basolateral intramembrane diffusion barrier and that the second extracellular loop of occludin plays a crucial role in the maintenance of epithelial cell polarity by the TJ.

Arabidopsisplasma membrane protein crucial for Ca2+influx and touch sensing in roots

Plants can sense and respond to mechanical stimuli, like animals. An early mechanism of mechanosensing and response is speculated to be governed by as-yet-unidentified sensory complexes containing a Ca(2+)-permeable, stretch-activated (SA) channel. However, the components or regulators of such complexes are poorly understood at the molecular level in plants. Here, we report the molecular identification of a plasma membrane protein (designated Mca1) that correlates Ca(2+) influx with mechanosensing in Arabidopsis thaliana. MCA1 cDNA was cloned by the functional complementation of lethality of a yeast mid1 mutant lacking a putative Ca(2+)-permeable SA channel component. Mca1 was localized to the yeast plasma membrane as an integral membrane protein and mediated Ca(2+) influx. Mca1 also increased [Ca(2+)](cyt) upon plasma membrane distortion in Arabidopsis. The growth of MCA1-overexpressing plants was impaired in a high-calcium but not a low-calcium medium. The primary roots of mca1-null plants failed to penetrate a harder agar medium from a softer one. These observations demonstrate that Mca1 plays a crucial role in a Ca(2+)-permeable SA channel system that leads to mechanosensing in Arabidopsis. We anticipate our findings to be a starting point for a deeper understanding of the molecular mechanisms of mechanotransduction in plants.

Viewpoint 6

Viewpoint 6

The role of AGEs for the pathogenesis of osteopenia in diabetes mellitus

Osteopenia was reported in patients with type I diabetes mellitus (DM). Also, serum levels of advanced glycation endproducts (AGEs) were reported to be elevated in DM. In this review, we showed the effect of AGEs on primary human osteoblasts, the precursor of human osteoclast like cells and parathyroid cells in culture. AGEs were made from incubating bovine serum albumin with glucose 6 phosphate for 8 weeks (AGEs-BSA). AGEs-BSA (1,000 microg/mL) showed inhibitory effect for human osteoblasts in terms of the productions of procolagen c propeptide and osteocalcin, which were regarded as bone forming parameters. On the other hand, the same concentration of AGEs-BSA inhibited the increase in the parathyroid hormone secretion from the cultured human parathyroid cells induced by low calcium medium concentration. AGEs-BSA increased time dependently intracellular calcium levels of HEK 293 cells, which expressed the wild type human calcium-sensing receptor. Also, AGEs-BSA increased IL-6 synthesis by primary human osteoblast. Our preliminary data also showed that AGEs-BSA increased osteoclast-like cells formation in number from the osteoclast precursor rich bone marrow cells. These data suggested the important role of AGEs for the pathogenesis of osteopenia in patients with DM through decreased bone formation and increased bone resorption.

A novel method of transfection of marine bacteria Pseudoalteromonas espejiana with deoxyribonucleic acid of bacteriophage PM2

DNA of bacteriophage PM2 is a convenient test object for studying DNA-damaging genotoxic agents. The extent of DNA damage can be estimated by the ability of damaged DNA for transfection of host cells, marine bacterium Pseudoalteromonas espejiana (Pae), str. BAL-31. The efficiency of transfection of Pae lines maintained for long periods without freezing was found to be very low upon the use of a widely accepted transfection method developed by van der Schans et al. (1971). Such cultures grown in a medium with 10 mM Ca2+ standard for Pae contained cell aggregates and exopolymer material. Pae was found to be capable of growing in a medium without the calcium supplement in the presence of chelator EGTA (low-calcium medium, LCM). After growth in LCM, cells did not aggregate, cultures lacked the activity of nuclease BAL, and transfection efficiency of cells grown in LCM drastically increased. Based on these results, a novel procedure of transfection with an efficiency of 2 x 10(4)-2 x 10(5) infectious centers per microgram of PM2 DNA was developed.

Opposing Roles of Zyxin/LPP ACTA Repeats and the LIM Domain Region in Cell-Cell Adhesion

Cadherins mediate cell-cell adhesion by linking cell junctions to actin networks. Although several actin regulatory systems have been implicated in cell-cell adhesion, it remains unclear how such systems drive cadherin-actin network formation and how they are regulated to coincide with initiation of adhesion. Previous work implicated VASP in assembly of cell-cell junctions in keratinocytes and the VASP-binding protein zyxin colocalizes with VASP at cell-cell junctions. Here we examine how domains in zyxin and its relative LPP contribute to cell-cell junction assembly. Using a quantitative assay for cell-cell adhesion, we demonstrate that zyxin and LPP function to increase the rate of early cell-cell junction assembly through the VASP-binding ActA repeat region. We also identify the LIM region of zyxin and LPP to be a regulatory domain that blocks function of these proteins. Deletion of the LIM domains drives adhesion and increases VASP level in detergent insoluble cadherin-actin. Dominant-negative zyxin/LPP mutants reduce the rate of adhesion, lower VASP levels in detergent-insoluble cadherin-actin networks, and allow for the accumulation of capping protein at cell-cell contacts. These data implicate the LIM domains of zyxin and LPP in regulating cell-cell junction assembly through VASP.

Low-calcium medium

Low-calcium medium

Isolation and characterization of a breast progenitor epithelial cell line with robust DNA damage responses

We report the establishment of a breast epithelial cell model that undergoes growth arrest at different stages of the cell cycle depending upon the DNA damaging agents encountered. Primary breast epithelial cells from normal reductive mammoplasty were grown in low-calcium culture medium. Free-floating cells under this condition were separated and used for establishment of the MCF-15 breast epithelial cell line. We found that MCF-15 breast epithelial cells display a superb response to different phases of the cell cycle arrest in response to various DNA damaging agents. Immunohistological analysis indicates that MCF-15 cells express cytokeratin 19, CD44, CXCR4, SDF-1, SPARC and vimentin. Although less than 5% of the MCF-15 cells expressed Muc-1 in culture, increased Muc-1 expression was observed in luminal epithelial cells along the newly formed lumen in xenografts. Furthermore, a small population of MCF-15 cells expressed estrogen receptor-alpha (ERalpha) in xenografts while ERalpha expression was not detected in monolayer culture. Therefore, the MCF-15 breast epithelial cell line possesses characteristics of breast progenitor cells and provides a good cell culture model for studying the response to DNA damage and the etiology of aggressive basal-like breast cancers.

SCCA2-transfected human keratinocytes show increased secretion of IL-1α and IL-6, but not of TNF-α

Psoriasis is a common skin disease characterized by epidermal hyperproliferation, capillary dilatation, and the presence of acute and chronic inflammatory cells in both the dermis and epidermis [1, 2]. A unifying hypothesis that accounts for the wide range of clinical characteristics of psoriasis highlights the role of cytokines in the etio-pathophysiology of this disease [8]. Epidermal cells, particularly keratinocytes, may play a central role in transmitting pro-inflammatory signals by the secretion of cytokines and growth factors [7]. The production of these factors, in particular IL-1 is usually very low, but can be induced significantly by various injurious agents, such as endotoxin, virus particles, tumor promoter or UV light [6, 7]. Both IL-1a and c are expressed in human keratinocytes, although the major isotype secreted is IL-1a [6]. IL-1 and TNF-a in their turn act with an autocrine mechanism on keratinocytes to induce the release of a cascade of other cytokines with pro-inflammatory or chemotactic activity (IL-6, IL-8) [3]. All of these mentioned cytokines have been detected in psoriatic lesions. In addition to these observations, Takeda et al. [11] reported that SCCA2, the member of serine proteinase inhibitor family (serpin) is the major inducible SCCA in psoriatic skin. They found that SCCA2 was expressed in stratum corneum, subset of spinous layer cells and dermal infiltrated cells [11]. The level of SCCA2 is significantly increased in the sera of psoriatic patients, compared with non-psoriatic individuals [4], and probably be due, at least in part, to the production of cytokines in psoriatic tissues. Moreover, recent study suggested that the expression of SCCA in keretinocytes may be participated in the growth of keratinocytes and their differentiation [11]. Since keratinocytes can secrete TNF-a, IL-1a, and IL-6, and since these molecules have been detected in psoriatic skin, the goal of this study was to test whether the expression of SCCA2 in human keratinocytes plays a role in modulating expression of these cytokines. Commercially available human epidermal keratinocytes (Kurabo, Japan) were grown in supplemented low calcium medium and were sub-cultured at 60–70% confluence to avoid differentiation. A SCCA2 construct consisting of the SCCA2 coding region cloned in to the p3XFLAG-myc-CMV-26 vector (Sigma) has been described elsewhere [12]. The vector [p3XFLAG-mycCMV-26 (Sigma)] without SCCA2 was transfected into the keratinocytes as a control. The SCCA2 construct or the control vector was transfected into human keratinocytes using lipofectamine reagent (Sigma) according to the manufacturer’s instructions. TNF-a, IL-1a, and IL-6 concentrations in the culture media were measured by using ELISA kits (R&D Systems Inc., USA) according to the manufacturer’s instructions. SCCA2 expression in transfected cell was determined by Western blot analysis using anti-SCCA antibody (Sigma). The statistical analysis was performed using Student’s t test, and P<0.05 was considered to be significant. The results are shown as mean ± standard deviation (SD). To show SCCA2 expression in cells transfected with the SCCA2 construct, cell lysates were subjected to Western blot analysis. SCCA2 expression was detected in cells transfected with the SCCA2 construct (Fig. 1a, lane 1) but not in cells transfected with vector alone (Fig. 1a, lane 2). Figure 2b shows a control blot using non-immunized mouse IgG. There is no conflict of interest in this manuscript.

Genotypic and Phenotypic Characteristics of Yersinia enterocolitica Isolated from the Surface of Chicken Eggshells Obtained in Argentina

The prevalence of Yersinia enterocolitica on chicken eggshell surfaces in San Luis, Argentina, was investigated. The pathogenic potential of recovered isolates was assessed by means of phenotypic virulence tests and the presence of the 72-kb pYV plasmid. Antimicrobial susceptibility was determined by the agar diffusion method. DNA digested with XbaI was analyzed by pulsed-field gel electrophoresis (PFGE), and relationships between genomic DNA profiles were established. Eight Y. enterocolitica B2 O:9 strains were recovered after enrichment, for a prevalence of 2.27%. All strains harbored the virulence pYV plasmid, bound Congo red, grew in a low-calcium medium, and autoagglutinated at 37°C. They lacked pyrazinamidase activity and did not hydrolyze esculin. These Y. enterocolitica strains were susceptible to amikacin, ciprofloxacin, chloramphenicol, and trimethoprim-sulfamethoxazole and were resistant to rifampin. According to the genomic DNA patterns obtained by PFGE, the isolates clustered into two groups, I and II. The highest similarity coefficient observed between Y. enterocolitica strains was 0.947. Microbiological controls on production stages of eggs and good culinary practices are necessary to reduce the risk of Y. enterocolitica infection for consumers.

Medium Calcium Concentration Determines Keratin Intermediate Filament Density and Distribution in Immortalized Cultured Thymic Epithelial Cells (TECs)

Isolation and culture of thymic epithelial cells (TECs) using conventional primary tissue culture techniques under conditions employing supplemented low calcium medium yielded an immortalized cell line derived from the LDA rat (Lewis [Rt1l] cross DA [Rt1a]) that could be manipulated in vitro. Thymi were harvested from 4-5-day-old neonates, enzymically digested using collagenase (1 mg/ml, 37 degrees C, 1 h) and cultured in low calcium WAJC404A medium containing cholera toxin (20 ng/ml), dexamethasone (10 nM), epidermal growth factor (10 ng/ml), insulin (10 mug/ml), transferrin (10 mug/ml), 2% calf serum, 2.5% Dulbecco's Modified Eagle's Medium (DMEM), and 1% antibiotic/antimycotic. TECs cultured in low calcium displayed round to spindle-shaped morphology, distinct intercellular spaces (even at confluence), and dense reticular-like keratin patterns. In high calcium (0.188 mM), TECs formed cobblestone-like confluent monolayers that were resistant to trypsinization (0.05%) and displayed keratin intermediate filaments concentrated at desmosomal junctions between contiguous cells. Changes in cultured TEC morphology were quantified by an analysis of desmosome/membrane relationships in high and low calcium media. Desmosomes were significantly increased in the high calcium medium. These studies may have value when considering the growth conditions of cultured primary cell lines like TECs.

Altered Gene Expression of Angiogenic Factors Induced by Calcium-Mediated Dissociation of Retinal Pigment Epithelial Cells

To examine the effect of loss of cell-cell contacts on the gene expression of vascular endothelial growth factor (VEGF) and other factors in primary culture of human retinal pigment epithelial (RPE) cells with real-time reverse transcription-PCR. The dissociation of postconfluent RPE cells was induced by calcium chelation, low-calcium medium, anti-E-cadherin, and anti-N-cadherin antibodies. Total RNA was isolated from the cultured RPE cells and reverse transcribed to cDNA. VEGF was quantified by real-time PCR with a fluorescence detector. VEGF isoforms were differentially measured by specific exon-spanning primers. Besides VEGF, the gene expression levels of some other growth factors were also examined in calcium-mediated dissociation. Disruption of cell-cell contacts of RPE cells was induced by calcium chelation and low-calcium medium, but not by anti-E-cadherin and anti-N-cadherin antibodies. Calcium-mediated dissociation of RPE cells significantly increased the gene expression levels of VEGF. The mRNA levels of VEGF increased by 6.3-fold on treatment with EGTA and by 4.7-fold in the low-calcium medium at 6 hours. Splice variants of VEGF showed the differential pattern of gene expression. Whereas the expression of VEGF(121) and VEGF(165) was upregulated on calcium-induced dissociation of RPE cells, that of VEGF(145) and VEGF(189) was unchanged. VEGF(206) was not detected. On calcium-induced dissociation, bFGF, IL-6, matrix metalloproteinase (MMP)-1, and placental growth factor (PlGF) were upregulated, whereas acidic (a)FGF and pigment epithelium-derived factor (PEDF) were both downregulated. The results show that loss of intercellular contacts promotes increased gene expression of VEGF and other angiogenic factors in human RPE cells.

β-Catenin/T-cell factor-mediated transcription is modulated by cell density in human bronchial epithelial cells

The embryonic Wnt/β-catenin (‘canonical’) pathway has been implicated in epithelial regeneration. To investigate the role of Wnt signal transduction in the airways, we characterised the expression of key pathway components in human bronchial epithelial cells (HBEC) and studied the influence of cell density on pathway activity, using sub-confluent cells in log-phase growth as a simple model of repairing epithelium. Primary HBEC and H292 bronchial epithelial cells were found to express TCF-4, TCF-3 and isoforms of LEF-1, transcription factors that are regulated by Wnt signalling. The cells also had the potential to respond to Wnt signalling through expression of several members of the Frizzled receptor family, including FZD-5 and -6. In confluent H292 cells, 20 mM lithium and 25% v/v Wnt-3a conditioned medium induced 4.5-fold ( p = 0.008) and 1.4-fold ( p = 0.006) increases in TOPflash activity, respectively. Under conditions of reduced cell density, TOPflash activity increased 1.8-fold ( p = 0.002) in association with increased nuclear localisation of hypophosphorylated (active) β-catenin and increased cell proliferation. This up-regulation in reporter activity occurred independently of EGF receptor activation and could not be recapitulated by use of low-calcium medium to disrupt cadherin-mediated cell–cell adhesion, but was associated with changes in FZD-6 expression. We conclude that reactivation of this embryonic pathway may play an important role in bronchial epithelial regeneration, and that modulation of Fzd-6 receptors may regulate Wnt signalling at confluence. Recognising that many chronic inflammatory disorders of the airways involve epithelial damage and repair, altered Wnt signalling might contribute to disease pathogenesis or progression.

1,25-Dihydroxyvitamin D 3 up-regulates the renal vitamin D receptor through indirect gene activation and receptor stabilization

Expression of the vitamin D receptor (VDR) in the kidney and intestine plays a major role in calcium homeostasis and the metabolism of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3). Calcium and 1,25(OH) 2D 3-mediated regulation of renal and duodenal VDR expression has been analyzed in vivo and the mechanisms responsible for the renal regulation have been studied in mouse kidney TCMK-1 cells. Vitamin D-deficient mice were maintained on diets containing either 0.02 or 0.47% calcium, with or without 50 ng of 1,25(OH) 2D 3 per day. Renal VDR levels were significantly higher in the vitamin D-deficient mice fed the 0.47% calcium diet vs. the calcium-restricted diet, and were increased 5-fold by 1,25(OH) 2D 3 when dietary calcium was present. The renal VDR transcript was expressed at a basal level in the absence of calcium or 1,25(OH) 2D 3; 50 ng of 1,25(OH) 2D 3 elevated renal VDR mRNA levels approximately 10-fold in the presence of calcium. Neither calcium nor 1,25(OH) 2D 3 had any significant effect on duodenal VDR or VDR mRNA expression. In TCMK-1 cells, 1,25(OH) 2D 3 increased receptor and VDR mRNA content in both low and adequate calcium medium. The 1,25(OH) 2D 3-mediated increase in VDR mRNA did not result from increased stability of the transcript. Further, the increase in mRNA was blocked by cycloheximide, indicating a requirement for protein synthesis and an indirect regulation of VDR transcription. Thus, both dietary serum calcium and 1,25-(OH) 2D 3 are required for VDR expression in kidney but not in intestine where neither is required. The 1,25-(OH) 2D 3 requirement can also be shown in TCMK-1 cells in vitro, while the calcium requirement was not found.

A novel sensor kinase–response regulator hybrid regulates type III secretion and is required for virulence in Pseudomonas aeruginosa

The type III secretion system (TTSS) of Pseudomonas aeruginosa is induced by contact with eukaryotic cells and by growth in low-calcium media. We have identified a protein, RtsM, that is necessary for expression of the TTSS genes in P. aeruginosa. RtsM possesses both histidine kinase and response regulator domains common to two-component signalling proteins, as well as a large predicted periplasmic domain and seven transmembrane domains. Deletion of rtsM resulted in a defect in production and secretion of the type III effectors. Northern blot analysis revealed that mRNAs encoding the effectors ExoT and ExoU are absent in the DeltartsM strain under TTSS-inducing conditions. Using transcriptional fusions, we demonstrated that RtsM is required for transcription of the operons encoding the TTSS effectors and apparatus in response to calcium limitation or to host cell contact. The operon encoding the TTSS regulator ExsA does not respond to calcium limitation, but the basal transcription rate of this operon was lower in deltartsM than in the wild-type parent, PA103. The defect in TTSS effector production and secretion of deltartsM could be complemented by overexpressing ExsA or Vfr, two transcriptional activators involved in TTSS regulation. DeltartsM was markedly less virulent than PA103 in a murine model of acute pneumonia, demonstrating that RtsM is required in vivo. We propose that RtsM is a sensor protein at the start of a signalling cascade that induces expression of the TTSS in response to environmental signals.

Towards optimization of an organotypic assay system that imitates human hair follicle-like epithelial-mesenchymal interactions

Human hair growth can currently be studied in vitro by the use of organ-cultured scalp hair follicles (HFs). However, simplified organotypic systems are needed for dissecting the underlying epithelial-mesenchymal interactions and as screening tools for candidate hair growth-modulatory agents. To optimize the design and culture conditions of previously published organotypic systems that imitate epithelial-mesenchymal interactions in the human HF as closely as possible. Continuous submerged organotypic 'sandwich' cultures were established. These consist of a pseudodermis (collagen I mixed with and contracted by human interfollicular dermal fibroblasts) on which one of two upper layers is placed: either a mixture of Matrigel basement membrane matrix (BD Biosciences, Bedford, MA, U.S.A.) and follicular dermal papilla fibroblasts (DPC), with outer root sheath keratinocytes (ORSK) layered on the top ('layered' system), or a mixture of Matrigel, DPC and ORSK ('mixed' system). Morphological and functional characteristics of these 'folliculoid sandwiches' were then assessed by routine histology, histomorphometry and immunohistochemistry. In both 'layered' and 'mixed' systems, the ORSK formed spheroid epithelial cell aggregates, which retained their characteristic keratin expression pattern (i.e. cytokeratin 6). In the 'mixed' sandwich model the size of the epithelial cell aggregates was smaller, but the numbers of ORSK were significantly higher than in the 'layered' model at day 14 in the culture. ORSK proliferated better in the 'mixed' than in the 'layered' sandwich system, regardless of the calcium or serum content of the media, whereas apoptosis of ORSK was lowest in the 'mixed' system in serum-free, low calcium medium. The kinetics of proliferation and apoptosis of DPC, which retained their characteristic expression of versican, were similar in both systems. However, proliferation and apoptosis of DPC were higher in the presence of serum and/or under high calcium conditions. Our results underscore the importance of structural design and medium composition for epithelial-mesenchymal interactions as they occur in the human HF. Specifically, we report a new organotypic submerged 'folliculoid sandwich' system with serum-free, low calcium medium and a mixture of interacting human DPC and ORSK, which offers several advantages over previously available assays. This system allows the standardized assessment of the effects of a test agent on the proliferation, apoptosis and key marker expression of human ORSK and DPC under substantially simplified in vitro conditions which approximate the in vivo situation.