Proliferation Of Human Intestinal Cells Research Articles

Systematic gene silencing identified Cryptosporidium nucleoside diphosphate kinase and other molecules as targets for suppression of parasite proliferation in human intestinal cells

Cryptosporidiosis is a major cause of diarrheal disease. The only drug approved for cryptosporidiosis has limited efficacy in high-risk populations. Therefore novel drugs are urgently needed. We have identified several enzymes as potential targets for drug development and we have optimized a rapid method to silence genes in Cryptosporidium. In this study, we knocked down expression of the four selected genes: Actin (Act), Apicomplexan DNA-binding protein (Ap2), Rhomboid protein 1 (Rom 1), and nucleoside diphosphate kinase (NDK). After gene silencing, we evaluated the role of each target on parasite development using in vitro models of excystation, invasion, proliferation, and egress. We showed that silencing of Act, Ap2, NDK, and Rom1 reduced invasion, proliferation, and egress of Cryptosporidium. However, silencing of NDK markedly inhibited Cryptosporidium proliferation (~70%). We used an infection model to evaluate the anticryptosporidial activity of ellagic acid (EA), an NDK inhibitor. We showed that EA (EC50 = 15–30 µM) reduced parasite burden without showing human cell toxicity. Here, we demonstrated the usefulness of a rapid silencing method to identify novel targets for drug development. Because EA is a dietary supplement already approved for human use, this compound should be studied as a potential treatment for cryptosporidiosis.

Human and bovine osteopontin from milk and recombinant human osteopontin may stimulate intestinal proliferation and immune functions via various mechanisms revealed by microarray analysis

Osteopontin (OPN) is a highly acidic phosphorylated glycoprotein, present at high concentration in human milk and low concentration in bovine milk and infant formula. Human OPN (hOPN) and bovine OPN (bOPN) have been demonstrated to be resistant to in vitro digestion. Thus, hOPN and bOPN may play important roles in intestinal and immunological development in infancy. hOPN, recombinant hOPN (rhOPN) and bOPN may function differently due to different post‐translational modifications and 63% amino acid identity between hOPN and bOPN. Therefore, we hypothesized that hOPN, rhOPN and bOPN are capable to stimulate intestinal proliferation and regulate immune functions via various mechanisms. BrdU assays showed that hOPN, rhOPN and bOPN significantly stimulated proliferation of human intestinal cells (HIEC). Compared with hOPN and rhOPN, a lower concentration was needed for proliferation effects exerted by bOPN. To screen for underlying mechanisms by which hOPN, rhOPN and bOPN stimulate intestinal proliferation, microarray assays (Illumina) were performed. Selected genes were verified by RT‐PCR with a high validation rate. Genes significantly regulated by hOPN, rhOPN, and bOPN were 243, 356, and 284 respectively of which only 50 genes were modulated by all. Genes tightly related to proliferation and immune function were significantly modified by hOPN, eg MAPK13, CCNE1, CdGAP, CXCL10, IL6ST and NFKB. Some of these genes were affected by rhOPN or bOPN, but not all. In summary, hOPN, rhOPN and bOPN are capable to stimulate intestinal proliferation and regulate immune functions but via various mechanisms.

Integrin‐linked kinase regulates migration and proliferation of human intestinal cells under a fibronectin‐dependent mechanism

Integrin-linked kinase (ILK) plays a role in integrin signaling-mediated extracellular matrix (ECM)–cell interactions and also acts as a scaffold protein in functional focal adhesion points. In the present study, we investigated the expression and roles of ILK in human intestinal epithelial cells (IECs) in vivo and in vitro. Herein, we report that ILK and its scaffold-function interacting partners, PINCH-1, α-parvin, and β-parvin, are expressed according to a decreasing gradient from the bottom of the crypt (proliferative/undifferentiated) compartment to the tip of the villus (non-proliferative/differentiated) compartment, closely following the expression pattern of the ECM/basement membrane component fibronectin. The siRNA knockdown of ILK in human IECs caused a loss of PINCH-1, α-parvin, and β-parvin expression, along with a significant decrease in cell proliferation via a loss of cyclin D1 and an increase in p27 and hypophosphorylated pRb expression levels. ILK knockdown severely affected cell spreading, migration, and restitution abilities, which were shown to be directly related to a decrease in fibronectin deposition. All ILK knockdown-induced defects were rescued with exogenously deposited fibronectin. Altogether, our results indicate that ILK performs crucial roles in the control of human intestinal cell and crypt–villus axis homeostasis—especially with regard to basement membrane fibronectin deposition—as well as cell proliferation, spreading, and migration. J. Cell. Physiol. 222: 387–400, 2010. © 2009 Wiley-Liss, Inc.

The human intestinal microbiome: a new frontier of human biology.

To analyze the vast number and variety of microorganisms inhabiting the human intestine, emerging metagenomic technologies are extremely powerful. The intestinal microbes are taxonomically complex and constitute an ecologically dynamic community (microbiota) that has long been believed to possess a strong impact on human physiology. Furthermore, they are heavily involved in the maturation and proliferation of human intestinal cells, helping to maintain their homeostasis and can be causative of various diseases, such as inflammatory bowel disease and obesity. A simplified animal model system has provided the mechanistic basis for the molecular interactions that occur at the interface between such microbes and host intestinal epithelia. Through metagenomic analysis, it is now possible to comprehensively explore the genetic nature of the intestinal microbiome, the mutually interacting system comprising the host cells and the residing microbial community. The human microbiome project was recently launched as an international collaborative research effort to further promote this newly developing field and to pave the way to a new frontier of human biology, which will provide new strategies for the maintenance of human health.

Biological activity of bovine milk on proliferation of human intestinal cells

To evaluate the bioactivity of bovine milk from different stages of lactation on human intestinal tissue, a human fetal small intestinal cell line was used as a model system. Milk samples representing six stages of lactation: days 1, 2-3, 6-7 and weeks 12 and 24 after parturition, 1 week before drying off, and milk-like secretion from two stages of the dry period: 7 weeks and 3-4 weeks before expected calving, were collected from 64 Holstein Friesian cows. The whey fraction of the milk or milk-like secretion was added to the culture medium in concentrations ranging from 0.078% to 10%. The growth-promoting activity of whey was measured by determining the incorporation of [3H]thymidine into DNA for the last 24 h of the culture period. Whey fractions from all six stages of lactation stimulated growth of intestinal cells. The growth-promoting activity of colostrum or milk significantly decreased within the first week after calving. The growth-promoting activity in mature milk increased gradually during lactation to reach a level significantly higher than that obtained with colostrum. The growth-promoting activity of whey from milk-like secretion collected after drying off was lower than that of colostrum. Whey from different stages of lactation contained significantly different concentrations of TGF-beta1 (0.5-27 ng/ml) and TGF-beta2 (12-1219 ng/ml). However, neither the differences in TGF-beta1 and TGF-beta2, nor the differences in IGF-I and IGF-binding proteins could fully explain the differences in growth-promoting activity of colostrums or milk from different stages of lactation, suggesting that other factors were also involved. The present study showed that bovine milk contained a number of biologically active components that affected growth and development of human intestinal tissue. The results showed that the growth-promoting activity of colostrum and milk was dependent on the stage of lactation in accordance with previous results obtained with mammary epithelial cells. The changes in growth-promoting activity with stage of lactation were probably related to changes in concentrations of several growth factors.

Regulated expression of the integrin α9β1 in the epithelium of the developing human gut and in intestinal cell lines: Relation with cell proliferation

The integrin alpha9beta1 is one of the recently identified integrins whose expression is restricted to specialized tissues. Its exact function is still unknown. In the present study, we have analyzed the expression of the alpha9 subunit in human fetal and adult small intestinal and colonic epithelia as well as in intestinal cell lines by indirect immunofluorescence, immunoprecipitation, Western blot, and Northern blot. In intact tissues, the antigen was restricted to the basolateral domain of epithelial cells in intestinal crypts at the fetal stage and was absent in the adult. The alpha9beta1 integrin was also detected in the intestinal cell lines HIEC-6 and Caco-2/15. The presence of alpha9beta1 in HIEC-6 was found to be consistent with their proliferative crypt-like status. In Caco-2/15 cells, the integrin was present at high levels in proliferating cells but was downregulated when cells cease to grow and undertake their differentiation. EGF treatment, which is known to maintain Caco-2/15 cells in a proliferative state, resulted in higher levels of alpha9 as compared to control cells. Taken together, these observations suggest a relation between integrin alpha9beta1 expression and proliferation in human intestinal cells.

Regulated expression of the integrin α9β1 in the epithelium of the developing human gut and in intestinal cell lines: Relation with cell proliferation

The integrin alpha9beta1 is one of the recently identified integrins whose expression is restricted to specialized tissues. Its exact function is still unknown. In the present study, we have analyzed the expression of the alpha9 subunit in human fetal and adult small intestinal and colonic epithelia as well as in intestinal cell lines by indirect immunofluorescence, immunoprecipitation, Western blot, and Northern blot. In intact tissues, the antigen was restricted to the basolateral domain of epithelial cells in intestinal crypts at the fetal stage and was absent in the adult. The alpha9beta1 integrin was also detected in the intestinal cell lines HIEC-6 and Caco-2/15. The presence of alpha9beta1 in HIEC-6 was found to be consistent with their proliferative crypt-like status. In Caco-2/15 cells, the integrin was present at high levels in proliferating cells but was downregulated when cells cease to grow and undertake their differentiation. EGF treatment, which is known to maintain Caco-2/15 cells in a proliferative state, resulted in higher levels of alpha9 as compared to control cells. Taken together, these observations suggest a relation between integrin alpha9beta1 expression and proliferation in human intestinal cells.