MicroRNA-216b inhibits heat stress-induced cell apoptosis by targeting Fas in bovine mammary epithelial cells

Heat stress affects milk yield and quality in lactating dairy cows in summer. Bovine mammary epithelial cells (bMECs) play a key role in milk secretion, and microRNAs (miRNAs) regulate numerous functions of bMEC. Previous reports have verified that miR-216b regulated cell apoptosis through repressing target genes in several cancer cells. So, our purpose was to explore the potential involvement of miR-216b in heat stress-induced cell apoptosis in bMECs. Firstly, the heat stress model was constructed and we found that apoptotic rates of bMECs significantly increased under heat stress. The expression of miR-216b, Bax mRNA, and caspase-3 mRNA was upregulated. However, Bcl-2 mRNA level was detected to differentially downregulated. Overexpression of miR-216b remarkably downregulated the expression of caspase-3 and Bax mRNA and protein, and the mRNA and protein level of Bcl-2 was increased. Inhibition of miR-216b increased the activity of caspase-3 and Bax, and the level of Bcl-2 was inhibited. Moreover, Fas was identified as a target gene of miR-216b through bioinformatic analysis and dual-luciferase reporter assay. Fas activity was significantly inhibited and enhanced respectively after transfecting miRNA mimics and inhibitor. Finally, inhibition of Fas via the small interfering RNA (siRNA) also inhibited cell apoptosis induced by heat stress. Taken together, our results indicated that miR-216b exerted as an anti-apoptotic effect under heat stress in bMECs by targeting Fas.     

Inhibitory effect of lactoferrin on hypertrophic differentiation of ATDC5 mouse chondroprogenitor cells

The skeleton is formed by two different mechanisms. In intramembranous ossification, osteoblasts form bone directly, whereas in endochondral ossification, chondrocytes develop a cartilage template, prior to osteoblast-mediated skeletogenesis. Lactoferrin is an iron-binding glycoprotein belonging to the transferrin family. It is known to promote the growth and differentiation of osteoblasts. In this study, we investigated the effects of bovine lactoferrin on the chondrogenic differentiation of ATDC5 chondroprogenitor cells. This mouse embryonic carcinoma-derived clonal cell line provides an in vitro model of chondrogenesis. Lactoferrin treatment of differentiating ATDC5 cells promoted cell proliferation in the initial stage of the differentiation process. However, lactoferrin treatment resulted in inhibition of hypertrophic differentiation, characterized by suppression of alkaline phosphatase activity, aggrecan synthesis and N-cadherin expression. This inhibitory effect was accompanied by sustained Sox9 expression, as well as increased Smad2/3 expression and phosphorylation, suggesting that lactoferrin regulates chondrogenic differentiation by up-regulating the Smad2/3-Sox9 signaling pathway.     

Identification of lactoferrin as an essential growth factor for human lymphocytic cell lines in serum-free medium

A serum-free medium supplemented with growth factor(s) was devised to grow human lymphocytic cell lines. The medium was developed using human lymphocytic cell line, Bri 7 cells. In the process of constructing the medium, human lactoferrin was found to be an essential growth factor for the cell line. Human lactoferrin has higher growth stimulatory activity than human transferrin, and was sensitive to heat. Long-term cultivation of the cells was achieved in the defined medium supplemented with human lactoferrin only. The defined medium specifically supported the growth of various other human B- and T-lymphocytic cell lines but not the growth of various mouse lymphocytic cell lines. In lactoferrin-supplemented medium, the growth of some human cell lines were further stimulated by the addition of a combination of insulin, ethanolamine and selenium, or another combination of 2-mercaptoethanol and the above three factors. Bovine lactoferrin could be substituted for human lactoferrin.     

Identification of lactoferrin as an essential growth factor for human lymphocytic cell lines in serum-free medium

A serum-free medium supplemented with growth factor(s) was devised to grow human lymphocytic cell lines. The medium was developed using human lymphocytic cell line, Bri 7 cells. In the process of constructing the medium, human lactoferrin was found to be an essential growth factor for the cell line. Human lactoferrin has higher growth stimulatory activity than human transferrin, and was sensitive to heat. Long-term cultivation of the cells was achieved in the defined medium supplemented with human lactoferrin only. The defined medium specifically supported the growth of various other human B- and T-lymphocytic cell lines but not the growth of various mouse lymphocytic cell lines. In lactoferrin-supplemented medium, the growth of some human cell lines were further stimulated by the addition of a combination of insulin, ethanolamine and selenium, or another combination of 2-mercaptoethanol and the above three factors. Bovine lactoferrin could be substituted for human lactoferrin.     

Casein gene expression in mouse mammary epithelial cell lines: dependence upon extracellular matrix and cell type

The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D line. None of the four clonal lines appeared to represent myoepithelial cells. The cell lines were examined for expression of beta-casein mRNA in the presence or absence of prolactin. The heterogeneous COMMA-D line, but none of the clonal lines, was induced by the presence of prolactin to produce significantly increased levels of beta-casein MRNA. The inducibility of beta-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. Individual matrix components of laminin, fibronectin, heparan sulfate, heparan, or hyaluronic acid were not effective as substrata for the induction of beta-casein mRNA. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types.     

Effect of lactoferrin on the growth of a human colon adenocarcinoma cell line--comparison with transferrin

Lactoferrin was examined for its effect on the growth of a human colon adenocarcinoma cell line (HT 29) in culture and its action was compared to that produced by transferrin and two different iron solutions (ferrous sulfate and ferric chloride). When transferrin was replaced by either iron solutions the cell grew in proportion to the quantity added and the maximal effect obtained was identical to that produced by transferrin alone. When transferrin was replaced by lactoferrin the cells were unable to proliferate for a long time. However, in the presence of low-concentration iron solutions, lactoferrin stimulated the cell growth, and the effect was more pronounced with the ferric chloride solution.     

Defensin γ-thionin from Capsicum chinense has immunomodulatory effects on bovine mammary epithelial cells during Staphylococcus aureus internalization

β-Defensins are members of the antimicrobial peptide superfamily that are produced in various species from different kingdoms, including plants. Plant defensins exhibit primarily antifungal activities, unlike those from animals that exhibit a broad-spectrum antimicrobial action. Recently, immunomodulatory roles of mammal β-defensins have been observed to regulate inflammation and activate the immune system. Similar roles for plant β-defensins remain unknown. In addition, the regulation of the immune system by mammalian β-defensins has been studied in humans and mice models, particularly in immune cells, but few studies have investigated these peptides in epithelial cells, which are in intimate contact with pathogens. The aim of this work was to evaluate the effect of the chemically synthesized β-defensin γ-thionin from Capsicum chinense on the innate immune response of bovine mammary epithelial cells (bMECs) infected with Staphylococcus aureus, the primary pathogen responsible for bovine mastitis, which is capable of living within bMECs. Our results indicate that γ-thionin at 0.1 μg/ml was able to reduce the internalization of S. aureus into bMECs (∼50%), and it also modulates the innate immune response of these cells by inducing the mRNA expression (∼5-fold) and membrane abundance (∼3-fold) of Toll-like receptor 2 (TLR2), as well as by inducing genes coding for the pro-inflammatory cytokines TNF-α and IL-1β (∼14 and 8-fold, respectively) before and after the bacterial infection. γ-Thionin also induces the expression of the mRNA of anti-inflammatory cytokine IL-10 (∼12-fold). Interestingly, the reduction in bacterial internalization coincides with the production of other antimicrobial products by bMECs, such as NO before infection, and the secretion into the medium of the endogenous antimicrobial peptide DEFB1 after infection. The results from this work support the potential use of β-defensins from plants as immunomodulators of the mammalian innate immune response.     

Effect of lactoferrin on the growth of a human colon adenocarcinoma cell line-comparison with transferrin

Summary Lactoferrin was examined for its effect on the growth of a human colon adenocarcinoma cell line (HT 29) in culture and its action was compared to that produced by transferrin and two different iron solutions (ferrous sulfate and ferric chloride). When transferrin was replaced by either iron solutions the cell grew in proportion to the quantity added and the maximal effect obtained was identical to that produced by transferrin alone. When transferrin was replaced by lactoferrin the cells were unable to proliferate for a long time. However, in the presence of low-concentration iron solutions, lactoferrin stimulated the cell growth, and the effect was more pronounced with the ferric chloride solution. This work was supported by Grants Inserum 817014 and LA CNRS 202.     

Developmental gene expression of lactoferrin in duodenum and effect of weaning age on gene expression of lactoferrin in piglets

Two experiments were conducted to evaluate duodenal gene expression of lactoferrin and effect of weaning age on mRNA expression of lactoferrin in piglets using semi-quantitative RT-PCR analysis. In experiment 1, a total of 15 female Duroc x Landrace x Yorkshire piglets of five groups, each group pigs at 1, 14, 28, 42 and 56 days of age were used to determine developmental gene expression of lactoferrin in duodenum. In experiment 2, a total of 18 female neonatal piglets were divided into three groups, which were weaned at 21, 28 and 35 days of age respectively. In each group, three piglets' duodena were sampled at 21, 28 and 35 days of age and the other three piglets' duodena were sampled 7 days after weaning in each group. The samples were collected for detecting the effect of weaning age on lactoferrin mRNA expression of piglets. The results show that lactoferrin mRNA levels decreased steadily in postnatal day 1-56. But only from day 28-42 (14 days after weaned), the levels of lactoferrin gene expression were decreased markedly (p < 0.05), and the difference of lactoferrin mRNA levels at other stages was not significant. This result suggested that weaning had an effect on gene expression of lactoferrin. The results of experiment 2 showed that when the piglets were weaned at 21-28 and 35 days of age respectively, the expression levels of lactoferrin were decreased by 77%, 53% and 59% at the seventh day after weaning. Our results showed that weaning significantly decreased lactoferrin mRNA expression of piglets.     

Characterization of a simian virus 40-transformed human podocyte cell line producing type IV collagen and exhibiting polarized response to atrial natriuretic peptide.

Biology of glomerular visceral epithelial cells ("podocytes") and their role in inflammatory process remain obscure, partly because of the lack of well-differentiated podocyte cultures. We have established a human cell line by transfecting with a replication-defective SV40 plasmid (pSVHB1), a primary culture of podocytes derived from an enriched preparation of unencapsulated glomeruli free of tubule and Bowman's capsule contaminants. Podocyte specificity of the primary culture was assessed by a dual immunomorphological and functional approach. The resulting cell line (HGVEC.SV1) was cloned and the clonal cells were adapted to hormonally defined medium supplemented with only 2% newborn bovine serum. Clone A4 has been exhibiting over 35 passages, a combination of markers unique to podocytes, including expression of vimentin, podocalyxin, ectoenzymes (CALLA antigen and mRNA), heparan-sulfate proteoglycans (molecular mass of core protein = 75 kDa), and production of type IV collagen (alpha 1 and alpha 5 chains) established by immunoprecipitation and Northern blot analysis. Cytokeratin was detected in rare cellular foci and the search of Von Willebrand factor was negative. This clonal cell line has been used to demonstrate: (1) that human podocytes are highly sensitive to atrial natriuretic peptide (ANP) which induced a dose-dependent increase in cGMP production (x20 at 0.5 microM ANP), and (2) that secretion of ANP-stimulated cGMP is dramatically polarized as 93% of extracellular cGMP were released in the apical medium when filter-grown HGVEC. SV1A4 cells were stimulated at their basal pole.     

Common and distinct factors regulate expression of mRNA for ETV5 and GDNF, Sertoli cell proteins essential for spermatogonial stem cell maintenance

Ets variant gene 5 (ETV5) and glial cell-derived neurotrophic factor (GDNF) are produced in Sertoli cells and required for maintenance and self-renewal of spermatogonial stem cells (SSCs) in mice. Fibroblast growth factors (FGFs) have been reported to stimulate Etv5 mRNA expression, and FSH was shown to stimulate Gdnf mRNA in Sertoli cell cultures, but there is no other information on factors that regulate these key Sertoli cell proteins necessary for stem cell maintenance. In this study, we investigated regulation of ETV5 and GDNF using the TM4 murine Sertoli cell line. FGF2 stimulated a time- and dose-dependent increase in Etv5 mRNA expression, with a maximal 8.3-fold increase at 6 h following 25 ng/ml FGF2 treatment. This FGF2 dose also stimulated Gdnf mRNA at 48 h. FGF2 effects on Etv5 and Gdnf mRNA were partially mediated through mitogen-activated protein kinase (MAPK) and phosphatidyl inositol 3-kinase (PI3K)-signaling cascades. Specific inhibitors of MAPK (PD98059) and PI3K (wortmannin) pathways reduced Etv5 and Gdnf mRNA expression in FGF2-treated cells. Epidermal growth factor (EGF) stimulated Etv5 mRNA but not Gdnf mRNA. TNFalpha and IL-1beta stimulated Gdnf mRNA, but had no effect on Etv5 mRNA. Other hormonal regulators of Sertoli cells such as testosterone, triiodothyronine and activin A did not affect Etv5 or Gdnf mRNA expression. Results with primary Sertoli cell cultures confirmed findings obtained with the TM4 cell line, validating the use of the TM4 model to examine regulation of Etv5 and Gdnf mRNA expression. In conclusion, we have identified common and unique pathways that regulate Etv5 and Gdnf mRNA in Sertoli cells, and FGFs are emerging as key regulators of the Sertoli cell proteins that control SSCs.     

The role of recombinant stem cell factor in early B cell development. Synergistic interaction with IL-7.

The cDNA for stem cell factor was recently isolated from Buffalo rat liver cells (BRL-3A) and recombinant rat stem cell factor produced from Escherichia coli (rrSCF164). rrSCF164 synergizes with rhIL-7 to stimulate pre-B clonal growth in agar culture of mouse bone marrow cells, and in this study we have characterized the role of rrSCF164 in B cell development. The combination of rrSCF164 plus rhIL-7 stimulated increased colony numbers compared with the sum of colonies stimulated by rrSCF164 and rhIL-7 alone. Also, increased cell proliferation per colony was stimulated by the combination of rrSCF164 plus rhIL-7 compared with rhIL-7 or rrSCF164 alone. The colonies formed with rrSCF164 plus rhIL-7 and rhIL-7 alone contained exclusively pre-B cells, which expressed B220 Ag and cytoplasmic mu-chain, but were negative for surface Ig expression. Morphological examination of the cells in the colonies showed blast-like characteristics. rrSCF164 alone and in combination with rhIL-7 stimulated generation of B220+ cells in liquid culture of B220- cells, whereas rhIL-7 alone had no stimulatory effect on B220- cells. Both stem cell factor mRNA and bioactivity were detected in a mouse bone marrow-derived stromal cell line, termed OZ-11. We propose that stem cell factor is a stromal-derived factor that synergizes with IL-7 to stimulate the proliferation and differentiation of pro-B cells to pre-B cells, which become responsive to IL-7 alone.     

An alpha-subunit-secreting cell line derived from a mouse thyrotrope tumor.

The anterior pituitary contains multiple distinct endocrine cell types that secrete individual hormones. To derive a pure cell culture population in which to study the regulation of the alpha-subunit of TSH free of other hormones and cell types, we have developed a clonal continuous cell line from the transplantable thyrotrope tumor MGH101A. This cell line expresses alpha-subunit mRNA, secretes alpha-subunit protein, and has maintained a stable phenotype for over 3 yr in culture. However, as is the case for the transplantable tumor from which they are derived, these cells do not express the beta-subunit of TSH or respond to TRH or thyroid hormone. We have used this cell line to investigate regulation of the alpha-subunit mRNA by the second messengers, cAMP and phorbol esters, and by glucocorticoids. Phorbol esters increase alpha-subunit mRNA levels significantly (3.5-fold), as does cAMP (1.8-fold). In contrast, glucocorticoids decrease mRNA levels from cAMP-induced or basal levels (2-fold). These cells should prove valuable for study of alpha-subunit gene expression in an isolated renewable clonal cell culture system.     

Recombinant human bone morphogenetic protein-2 stimulates osteoblastic maturation and inhibits myogenic differentiation in vitro

The in vitro effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on osteogenic and myogenic differentiation was examined in two clonal cell lines of rat osteoblast-like cells at different differentiation stages, ROB-C26 (C26) and ROB-C20 (C20). The C26 is a potential osteoblast precursor cell line that is also capable of differentiating into muscle cells and adipocytes; the C20 is a more differentiated osteoblastic cell line. Proliferation was stimulated by rhBMP-2 in C26 cells, but inhibited in C20 cells. rhBMP-2 greatly increased alkaline phosphate (ALP) activity in C26 cells, but not in C20 cells. The steady-state level of ALP mRNA was also increased by rhBMP-2 in C26 cells, but not in C20 cells. Production of 3',5'-cAMP in response to parathyroid hormone (PTH) was dose-dependently enhanced by adding rhBMP-2 in both C26 and C20 cells, though the stimulatory effect was much greater in the former. There was neither basal expression of osteocalcin mRNA nor its protein synthesis in C26 cells, but they were strikingly induced by rhBMP-2 in the presence of 1 alpha,25-dihydroxyvitamin D3. rhBMP-2 induced no appreciable changes in procollagen mRNA levels of type I and type III in the two cell lines. Differentiation of C26 cells into myotubes was greatly inhibited by adding rhBMP-2. The inhibitory effect of rhBMP-2 on myogenic differentiation was also observed in clonal rat skeletal myoblasts (L6). Like BMP-2, TGF-beta 1 inhibited myogenic differentiation. However, unlike BMP-2, TGF-beta 1 decreased ALP activity in both C26 and C20 cells. TGF-beta 1 induced neither PTH responsiveness nor osteocalcin production in C26 cells, but it increased PTH responsiveness in C20 cells. These results clearly indicate that rhBMP-2 is involved, at least in vitro, not only in inducing differentiation of osteoblast precursor cells into more mature osteoblast-like cells, but also in inhibiting myogenic differentiation.     

An evidence of stromal cell populations functionally linked with epithelial cell populations in the mouse oviduct

The oviductal epithelium consists of two major cell populations, secretory cells and cilial cells. In a previous report, we established clonal cell lines from the epithelium and stroma of an oviduct which allowed us to analyze stromal contribution to epithelial functions. Three stromal cell lines were co-cultured in separated apparatus with 3 epithelial cell lines, respectively. Two stromal cell lines preferentially stimulated mogp-1 expression on secretory cells and the stimulation was additive with estrogen. The lines had no effect on cilial cells. One stromal cell line preferentially stimulated foxj1 expression on cilial cells and the stimulation relieved suppression by estrogen. The line had no effect on secretory cells. Experiments with conditioned media of the stromal cells confirmed the results of co-culture experiments, suggesting that the oviductal stroma contains multiple cell populations preferentially regulating or modulating specific cell populations of the epithelium via diffusible factors.     

Differential expression and estrogen response of lactoferrin gene in the female reproductive tract of mouse,rat, and hamster

Lactoferrin, an iron-binding glycoprotein, kills bacteria and modulates inflammatory and immune responses. Presence of lactoferrin in the female reproductive tract suggests that the protein may be part of the mucosal immune system and act as the first line of defense against pathogenic organisms. We have discovered that lactoferrin is a major estrogen-inducible protein in the uterus of immature mice and is up-regulated by physiological levels of estrogen during proestrous in mature mice. In the present study, we examined lactoferrin gene expression and its response to estrogen stimulation in the female reproductive tract of several strains of immature mouse, rat, and hamster. The lactoferrin expression in the cycling adult female rat was also evaluated. Lactoferrin gene polymorphism exists among the different mouse strains. In the three inbred mouse strains studied, lactoferrin gene expression is stimulated by estrogen in the immature uterus, although it is less robust than in the outbred CD-1 mouse. We found that the lactoferrin gene is constitutively expressed in the epithelium of the vagina and the isthmus oviduct; however, it is estrogen inducible in the uterus of immature mice and rats. Furthermore, lactoferrin is elevated in the uterine epithelium of the mature rat during the proestrous and estrous stages of the estrous cycle. Estrogen stimulation of lactoferrin gene expression in the reproductive tract of an immature hamster is limited to the vaginal epithelium. The present study demonstrates differential expression and estrogen responsiveness of the lactoferrin gene in different regions of the female rodent reproductive tract and variation among the rodent species studied.