Mineralocorticoid receptors in the epithelial cells of human colon and ileum

Specific binding of [3H]aldosterone to the cytosolic fraction of epithelial cells was studied in the human colon and terminal ileum. Analysis of [3H]aldosterone binding to the epithelial cells of ascending colon, caecum and ileum as a function of [3H]aldosterone concentration revealed only one class of specific receptors with an affinity constant of about 2 nmol/l. [3H]aldosterone binding was approximately the same in the sigmoid, descending and transverse colon and in the caecum, but slightly lower in the ascending colon and ileum. The specificity of the [3H]aldosterone binding was the same along the colon. The relative order of potency in inhibiting [3H]aldosterone binding was: aldosterone = SC 26304 = dexamethasone much greater than dihydrotestosterone greater than estradiol = RU 26988.     

Insulin receptors and insulin effects on type II alveolar epithelial cells

Type II alveolar epithelial cells (pneumocytes) were isolated to purity from adult rabbits and analyzed for the presence of cell surface insulin receptors and for effects of insulin on cells. Assays were performed on cells cultured for 24 h in Eagle's minimum essential medium. Insulin binding to cells in culture approached a steady-state level by 180 min at 15 degrees C and remained constant for at least 1 h. Competition experiments using native insulin, proinsulin and desoctapeptide supported specificity of binding. Scatchard analysis of binding revealed a class of high-affinity receptors with Kd = 1.5 X 10(-10) M and a low-affinity component with Kd = 4 X 10(-9) M. The number of receptors was estimated at 2000-4000/cell. Insulin added to cell cultures of type II pneumocytes in concentrations from 5 X 10(-11) to 5 X 10(-8) M resulted in a dose-related increase in uptake of 2-deoxyglucose by cells. Insulin also stimulated the incorporation of choline and glucose into phosphatidylcholine and disaturated phosphatidylcholine.     

Identification and characterization of insulin receptors on foetal-mouse brain-cortical cells.

The occurrence of insulin receptors was investigated in freshly dissociated brain-cortical cells from mouse embryos. By analogy with classical insulin-binding cell types, binding of 125I-insulin to foetal brain-cortical cells was time- and pH-dependent, only partially reversible, and competed for by unlabelled insulin and closely related peptides. Desalanine-desasparagine-insulin, pig proinsulin, hagfish insulin and turkey insulin were respectively 2%, 4%, 2% and 200% as potent as bovine insulin in inhibiting 125I-insulin binding to brain-cortical cells, which corresponds to their relative biological potencies in classical insulin-target cells; no competition was observed with glucagon and nerve growth factor, even at high concentrations. Scatchard analysis of competitive-binding data resulted in curvilinear plots with a high-affinity binding of Ka = 3.6 X 10(8) M-1. Insulin binding to foetal brain-cortical cells differed, however, in two distinct aspects from that to classical insulin-binding cell types. Firstly, dilution of 125I-insulin-bound cells in the presence of unlabelled insulin did not accelerate dissociation of the labelled hormone. Secondly, exposure of brain-cortical cells to insulin before the binding assay enhanced insulin binding, suggesting up-regulation of insulin receptors in response to insulin. In conclusion, foetal-mouse brain-cortical cells bear specific binding sites for insulin. Their insulin receptor shows a marked specificity and affinity for insulin, but differs in at least two properties from most classical insulin receptors. These differences in hormone-receptor interaction could reflect structural differences between insulin receptors on embryonic and differentiated cells.     

Identification and characterization of a diamine exporter in colon epithelial cells

SLC3A2, a member of the solute carrier family, was identified by proteomics methods as a component of a transporter capable of exporting the diamine putrescine in the Chinese hamster ovary (CHO) cells selected for resistance to growth inhibition by high exogenous concentrations of putrescine. Putrescine transport was increased in inverted plasma membrane vesicles prepared from cells resistant to growth inhibition by putrescine compared with transport in inverted vesicles prepared from non-selected cells. Knockdown of SLC3A2 in human cells, using short hairpin RNA, caused an increase in putrescine uptake and a decrease in arginine uptake activity. SLC3A2 knockdown cells accumulated higher polyamine levels and grew faster than control cells. The growth of SLC3A2 knockdown cells was inhibited by high concentrations of putrescine. Knockdown of SLC3A2 reduced export of polyamines from cells. Expression of SLC3A2 was suppressed in human HCT116 colon cancer cells, which have an activated K-RAS, compared with their isogenic clone, Hkh2 cells, which lack an activated K-RAS allele. Spermidine/spermine N(1)-acetyltransferase (SAT1) was co-immunoprecipitated by an anti-SLC3A2 antibody as was SLC3A2 with an anti-SAT1 antibody. SLC3A2 and SAT1 colocalized on the plasma membrane. These data provide the first molecular characterization of a polyamine exporter in animal cells and indicate that the diamine putrescine is exported by an arginine transporter containing SLC3A2, whose expression is negatively regulated by K-RAS. The interaction between SLC3A2 and SAT1 suggests that these proteins may facilitate excretion of acetylated polyamines.     

Direct protective action of epidermal growth factor on isolated gastric mucosal surface epithelial cells.

Epidermal growth factor (EGF) protects gastric mucosa against acute injury produced by a variety of damaging agents, but the mechanism of its protective action is not clear. Since the surface epithelial cells (SEC) are important component of gastric mucosal defence, we studied whether EGF may directly protect isolated gastric SEC against ethanol injury in vitro, in condition independent of systemic factors and whether endogenous prostaglandins may play a role in EGF's protective action. The isolated SEC from rat gastric mucosa were preincubated in medium only, or medium containing 0.0001-10.0 micrograms/ml of h-rEGF for 15 minutes, and incubated with 8% ethanol for 1 hour. In another study the above experiment was repeated but cells were pretreated with 10(-4) or 10(-5) M indomethacin before EGF treatment. The cell viability was assessed by fast green exclusion test. Incubation of SEC with 8% ethanol significantly reduced SEC cell viability to 50 +/- 2%: EGF 0.1 or 1.0 microgram/ml significantly reduced ethanol induced damage (cell viability 59 +/- 3 and 62 +/- 3% respectively). Pretreatment with 10(-4) M indomethacin (the dose which does not affect SEC viability but inhibit PGE2 and PGI2 generation), significantly reduced protective action of EGF against 8% ethanol injury. EGF 1.0 and 10.0 micrograms/ml alone without ethanol increased PGE2 and 6 keto PGF1 alpha generation by SEC. These studies demonstrated: 1) EGF is able to protect gastric surface epithelial cells directly without mediation by systemic factors. 2) EGF induced protection of SEC may in part be mediated by prostaglandins.     

Endocrine cells in the rabbit colon mucosal epithelium

The epithelium of the rabbit colon was studied by light and electron microscopy. The highest number of endocrinocytes in colon are observed in terminal parts of colon, i.e. in a distal part of appendix (135 +/- 15 cells/mm2) and in rectum (142 +/- 20), to decrease in the ileocaeal region (caecum proxinmal part--39 +/- 9, colon proximal part--56 +/- 9), where the least number of cells was marked. Agrentaffin cells (EC) number the same way, however, with a weaker difference in the number of cells between terminal departments and ileocaeal region. An electron microscope study of mucosal epithelium of the colon enabled us to identify 5 types of endocrinocytes. I-III types: EC-, D- and L-cells. IV and V are seldom met types, the same way as the "mixed" cells have been indentified. Whose cytoplasm simultaneously contained both mucous and endocrine granules. The received data show a certain degree of similarity in the endocrine apparatus of the rabbit with that of humans, although essential differences exists in regards of the appendix pattern.     

Identification of S-nitrosylated proteins after chronic exposure of colon epithelial cells to deoxycholate

Apoptosis resistance, a condition favoring genomic instability, is associated with higher risk of colorectal cancer. Deoxycholate (DOC) is a hydrophobic bile salt found in high concentrations in colon cancer patients, and induces apoptosis in cultured colonic cells and ex vivo in colonic biopsies. We showed previously that the chronic exposure of colon cancer cells to increasing concentrations of DOC leads to apoptosis resistance, and the suggested mechanism involves oxidative/nitrosative stress. Nitric oxide (NO) is a key signaling molecule that regulates cell function in a variety of physiologic and pathophysiologic states. In part, NO exerts its actions by S-nitrosylation of target thiols, and several proteins are regulated through this PTM, including the caspases, the main effectors of apoptosis. Here, we performed a proteomics study in the DOC-induced apoptosis-resistant colon cell line, HCT-116RC. Its profile of S-nitrosylated proteins was compared to a control cell line not exposed to DOC. Eighteen differentially S-nitrosylated proteins were identified in the HCT-116RC cell line, 14 of these are novel targets of S-nitrosylation not previously reported. These proteins include cytoskeletal and signaling proteins, metabolic enzymes, chaperones, and redox- and differentiation-related proteins. These results broaden our knowledge of potential signal transduction pathways that may lead to the development of new biomarkers and therapy targets.     

Adherence of Candida to mucosal epithelial cells

Adherence abilities of 45 Candida strains to human buccal and vaginal epithelial cells in vitro were tested in two media: 0.9% saline and phosphate buffer. Candida albicans cells adhered more strongly to epithelial cells than fungal cells of other Candida species. These findings were statistically significant according to Mann-Whitney's "U" test with buccal epothelial cells in both of the test media and with vaginal cells in saline only.     

Extracellular HSP70 binding to surface receptors present on antigen presenting cells and endothelial/epithelial cells

Extracellular HSP70 has been found to participate in both innate and adaptive immune responses. However, little is known about the molecular mechanisms that mediate this process. Previous reports suggest that HSP70 interacts with antigen presenting cells (APC) through a plethora of surface receptors. In this study, we have examined the relative binding of potential HSP70 receptors and found high affinity binding to LOX-1 but not other structures with a role in HSP70-APC interactions such as LRP/CD91, CD40, TLR2, TLR4 or another c-type lectin family member (DC-SIGN) closely related to LOX-1. In addition to APC, HSP70 can avidly bind to non-APC cell lines, especially those from epithelial or endothelial background.     

Colonic epithelial cells express specific ligands for mucosal macrophage immunosuppressive receptors siglec-7 and -9

Immune cells are known to express specific recognition molecules for cell surface glycans. However, mechanisms involved in glycan-mediated cell-cell interactions in mucosal immunity have largely been left unaccounted for. We found that several glycans preferentially expressed in nonmalignant colonic epithelial cells serve as ligands for sialic acid-binding Ig-like lectins (siglecs), the immunosuppressive carbohydrate-recognition receptors carried by immune cells. The siglec ligand glycans in normal colonic epithelial cells included disialyl Lewis(a), which was found to have binding activity to both siglec-7 and -9, and sialyl 6-sulfo Lewis(x), which exhibited significant binding to siglec-7. Expression of these siglec-7/-9 ligands was impaired upon carcinogenesis, and they were replaced by cancer-associated glycans sialyl Lewis(a) and sialyl Lewis(x), which have no siglec ligand activity. When we characterized immune cells expressing siglecs in colonic lamina propriae by flow cytometry and confocal microscopy, the majority of colonic stromal immune cells expressing siglec-7/-9 turned out to be resident macrophages characterized by low expression of CD14/CD89 and high expression of CD68/CD163. A minor subpopulation of CD8(+) T lymphocytes also expressed siglec-7/-9. Siglec-7/-9 ligation suppressed LPS-induced cyclooxygenase-2 expression and PGE(2) production by macrophages. These results suggest that normal glycans of epithelial cells exert a suppressive effect on cyclooxygenase-2 expression by resident macrophages, thus maintaining immunological homeostasis in colonic mucosal membranes. Our results also imply that loss of immunosuppressive glycans by impaired glycosylation during colonic carcinogenesis enhances inflammatory mediator production.     

Proliferative effects of insulin analogues on mammary epithelial cells

Structural modification of insulin results in the generation of insulin analogues that show altered binding affinities to the insulin receptor and/or IGF-I receptor. As a consequence these insulin analogues may have increased mitogenic potency. Nine benign or malignant human mammary epithelial cells, which show different insulin receptor and IGF-I receptor expression patterns, were studied regarding mitogenicity of insulin and insulin analogues. Only insulin glargine showed a significantly higher proliferative effect on MCF-7 breast cancer cells compared to regular insulin among a panel of short- or long-acting insulin analogues, that are in clinical use.     

Characterization of insulin receptors in isolated epithelial cells of rat ventral prostate: effect of fasting

Insulin receptors have been characterized in rat prostatic epithelial cells by using [125I]insulin and a variety of physicochemical conditions. The binding data at equilibrium (2 h at 15 degrees C) could be interpreted in terms of two populations of insulin receptors: a class of receptors with high affinity (Kd = 2.16 nM) and low binding capacity (28.0 fmol mg-1 protein), and another class of receptors with low affinity (Kd = 0.29 microM) and high binding capacity (1.43 pmol mg-1 protein). Proinsulin exhibited a 63-fold lower affinity than insulin for binding sites whereas unrelated peptides were ineffective. The specific binding of insulin increased by about 50 per cent after 96 h of fasting; this increase could be explained by an increase of both the number of the high affinity-low capacity sites and the affinity of the low affinity-high capacity sites. These results together with previous studies on insulin action at the prostatic level strongly suggest that insulin may exert a physiological role on the prostatic epithelium.     

ER-annulate lamellar associations of mucosal epithelial cells of the rat jejunum

Summary Mucosal cells of the rat jejunum vary greatly in the relative abundance and kind of endoplasmic reticulum (ER). Many cells have few rough surfaced cisternal ER elements; these cells frequently are located in basal villous regions and generally are less differentiated than those near apices. Profiles of rough ER cisternae were often encountered in stacks of 3–4 parallel units in apical cells. A number of differentiating basal cells were found to contain ER-annulate lamellar associations arranged similarly to the ER stacks of apical cells. The annulate lamellae of this complex resemble those described for other rapidly differentiating or embryonic cells and may be derived from the nuclear envelope. They could be involved in the formation of the ER.Supported by Grants (GB-19111 and GU-3161) from the National Science Foundation     

Identification of epidermal growth factor-responsive genes in normal rat ovarian surface epithelial cells

Alteration in epidermal growth factor receptor (EGFR) family signaling is among the most frequently implicated effectors of human oncogenesis. Overexpression of members of this family of receptors has often been detected in many epithelial tumors and is believed to be associated with an overall poor prognosis in patients with cancer. Therefore, we hypothesized that identification of potential EGF target genes in normal cells will provide a basis for unbiased genetic analysis of this signaling pathway in cancer. We utilized Atlas Rat 1.2 nylon cDNA arrays (Clontech) to determine gene expression changes in normal rat ovarian surface epithelial (ROSE) cells following EGF treatment. The results indicate activation of genes involved in a wide variety of cellular mechanisms, including regulation of cell cycle and proliferation, apoptosis, and protein turnover. In addition, using an in vitro model of ovarian cancer, we demonstrated that malignant transformation of ROSE cells resulted in alteration of downstream effectors of the EGFR pathway, as exemplified by aberrant expression of p66Shc, c-Jun, c-Myc, c-Fos, Lot1, p21Cip/Waf, and cdc25A. These data suggest that knowledge of the downstream genetic lesions, which may result in loss of growth factor requirement of the affected cells, will be crucial for the selection of the EGFR pathway as an effective target for cancer therapy.     

Phenotypic changes and gene expression in human colon mucosal epithelial cells upon transfection of a SV40 DNA-GPT recombinant

Summary Phenotypic changes (increased longevity, decreased growth factor requirements, altered cell surface features, growth in semisolid agarose, and SV40 T antigen expression) suggesting in vitro transformation were displayed by human normal colon mucosal epithelial cells transfected with pSV3gpt, a pBR322 recombinant containing the SV40 “early” T antigen coding region and the dominant selectable marker bacterial gene, xanthine-guanine phosphoribosyltransferase. In contrast, control cultures which received neither DNA nor the recombinatn pSV2gpt (which is identical to pSV3gpt but lacks the SV40 T antigen region) were not phenotypically altered.     

Long-term culture of epithelial cells from the normal rat colon

Summary Serial passage cultures of colonic epithelial cells from young rats have been maintained for more than 6 months in Eagle's minimum essential medium buffered with HEPES (25 mM) and supplemented with 2.5% fetal bovine serum, 0.5 μg/ml insulin, 5.0 μg/ml transferrin, and antibiotics. The cells proliferated in this medium with a population doubling time of approximately 53 h. The cells retained differentiated morphology as evidenced by secretory activity and the presence of secretory granules, microvilli, tonofilaments, and desmosomal junctions. Further cells at the fourth passage had normal karyotypes with 42 chromosomes and exhibited anchorage dependent growth. High concentrations of fetal bovine serum (10 to 15%) exerted toxic effects on the colonic epithelial cell cultures. Supported by National Cancer Institute Contract N01-CP-75914.     

Identification of a novel antigen on the apical surface of rat alveolar epithelial type II and Clara cells

Here we describe a monoclonal antibody (MMC4) that recognizes a novel antigen on the apical surface of rat alveolar epithelial type II and Clara cells in the lung, proximal tubule epithelial cells in the kidney, and villus epithelial cells in the small intestine. Biochemical analysis showed that the MMC4 antigen was sensitive to heating and proteinase K digestion and that it is distributed in the detergent-rich phase after Triton X-114 phase separation. These data suggest that the MMC4 antigen is an integral membrane protein. Glycerol gradient sedimentation identified two forms of the MMC4 antigen: one with a sedimentation coefficient of 10.1 and one with a sedimentation coefficient of 1.66, suggesting that the antigen may be part of a multiprotein complex. During rat development (fetal day 16 to adult), the MMC4 antigen increased 12-fold in the lung and 200-fold in the kidney. In the intestine, the MMC4 antigen increased 150-fold by neonatal day 1 and then decreased to adult values. Our data demonstrate that the MMC4 antigen is unlike known type II cell- and Clara cell-associated proteins. The MMC4 monoclonal antibody will be useful as a marker of epithelial cell phenotype in development and injury studies.     

Immunophysiology of epithelial cells and pattern-recognition receptors

Many types of pattern-recognition receptors, including the group of signaling Toll-like receptors, have been found in epithelial cells. They serve for recognizing microorganisms, which induces the activation of epithelial cells followed by production of chemokines and antimicrobial substances. On the one hand, this attracts macrophages, neutrophils, and other cells of innate immunity eliminating microorganisms to the epithelial locus. The same pattern-recognition receptors are involved in cell activation resulting in the production of cytokines that trigger the controlled activation of adaptive-immunity cells. This leads to the formation of anti-bodies or cytotoxic T lymphocytes. On the other hand, activated epithelial cells produce an array of antimicrobial substances suppressing enteropathogenic microflora inhabiting external epithelial spaces. Conversely, local tolerance is developed with respect to predominant microflora of epithelial tracts. This tolerance encompasses both innate and adaptive immunities of subepithelial spaces. Although much remains to be understood regarding the mechanisms of its formation and maintenance, it is obvious that epithelial cells play an important role in these processes. Thus, epithelial cells “conduct” immune responses to both pathogenic microorganisms penetrating the host body and microorganisms inhabiting epithelial cellular spaces. The maintenance of the optimal composition of the microbial biocenosis of the human body is a function of its immune system.