Enteral nutrient deprivation in patients leads to a loss of intestinal epithelial barrier function.

OBJECTIVE: To investigate the effect of nutrient withdrawal on human intestinal epithelial barrier function (EBF). We hypothesized that unfed mucosa results in decreased EBF. This was tested in a series of surgical small intestinal resection specimens. DESIGN: Small bowel specifically excluding inflamed tissue, was obtained from pediatric patients (aged 2 days to 19 years) undergoing intestinal resection. EBF was assessed in Ussing chambers for transepithelial resistance (TER) and passage of fluorescein isothiocyanate (FITC)-dextran (4 kD). Tight junction and adherence junction proteins were imaged with immunofluorescence staining. Expression of Toll-like receptors (TLR) and inflammatory cytokines were measured in loop ileostomy takedowns in a second group of patients. RESULTS: Because TER increased with patient age (P < .01), results were stratified into infant versus teenage groups. Fed bowel had significantly greater TER versus unfed bowel (P < .05) in both age populations. Loss of EBF was also observed by an increase in FITC-dextran permeation in enteral nutrient-deprived segments (P < .05). Immunofluorescence staining showed marked declines in intensity of ZO-1, occludin, E-cadherin, and claudin-4 in unfed intestinal segments, as well as a loss of structural formation of tight junctions. Analysis of cytokine and TLR expression showed significant increases in tumor necrosis factor (TNF)-α and TLR4 in unfed segments of bowel compared with fed segments from the same individual. CONCLUSION: EBF declined in unfed segments of human small bowel. This work represents the first direct examination of EBF from small bowel derived from nutrient-deprived humans and may explain the increased incidence of infectious complications seen in patients not receiving enteral feeds.

Stimulation of intestinal growth and function with DPP4 inhibition in a mouse short bowel syndrome model.

Glucagon-like peptide-2 (GLP-2) has been shown to be effective in patients with short bowel syndrome (SBS), but it is rapidly inactivated by dipeptidyl peptidase IV (DPP4). We used an orally active DPP4 inhibitor (DPP4-I), MK-0626, to determine the efficacy of this approach to promote adaptation after SBS, determined optimal dosing, and identified further functional actions in a mouse model of SBS. Ten-week-old mice underwent a 50% proximal small bowel resection. Dose optimization was determined over a 3-day post-small bowel resection period. The established optimal dose was given for 7, 30, and 90 days and for 7 days followed by a 23-day washout period. Adaptive response was assessed by morphology, intestinal epithelial cell (IEC) proliferation (proliferating cell nuclear antigen), epithelial barrier function (transepithelial resistance), RT-PCR for intestinal transport proteins and GLP-2 receptor, IGF type 1 receptor, and GLP-2 plasma levels. Glucose-stimulated sodium transport was assessed for intestinal absorptive function. Seven days of DPP4-I treatment facilitated an increase in GLP-2 receptor levels, intestinal growth, and IEC proliferation. Treatment led to differential effects over time, with greater absorptive function at early time points and enhanced proliferation at later time points. Interestingly, adaptation continued in the group treated for 7 days followed by a 23-day washout. DPP4-I enhanced IEC proliferative action up to 90 days postresection, but this action seemed to peak by 30 days, as did GLP-2 plasma levels. Thus DPP4-I treatment may prove to be a viable option for accelerating intestinal adaptation with SBS.

Distraction-induced intestinal growth: the role of mechanotransduction mechanisms in a mouse model of short bowel syndrome.

Novel strategies are needed to address the problem of patients with short bowel syndrome. We previously demonstrated a three-fold lengthening of pig bowel after 2 weeks of applied distractive forces, but we have not elucidated the mechanisms facilitating this growth. We used a mouse model of distraction-induced enterogenesis. High molecular weight polyethylene glycol (PEG) osmotically stretched an isolated small bowel segment (PEG-stretch). Significant increases in villus height and crypt depth and in intestinal epithelial cell length and numbers suggested epithelial remodeling in addition to proliferation during enterogenesis. LC-MS/MS analysis showed a two-fold upregulation of α-actinin-1 and -4. We also demonstrated that p-focal adhesion kinase (FAK), FAK, α-actinin, and Rac1 were significantly upregulated and that F-actin was relocalized in PEG-stretch versus controls. Blockade of the phosphotidyl inositol 3' kinase pathway failed to influence the increase in proliferation or decline in apoptosis after stretch, suggesting alternative signaling pathways are used, including MEK and P38MAPK, which were both upregulated during enterogenesis. Our data suggests that several known mechanotransduction pathways drive distraction-induced enterogenesis.

Directed trans-differentiation of thymus cells into parathyroid-like cells without genetic manipulation.

Replacement of a diseased organ with an autologously derived tissue is an ideal therapy for some medical problems. However, it is difficult to recreate many adult human tissues in vitro due to the functionally necessary architecture of most organs and the lack of understanding of methods to direct the development of the organ of interest. The parathyroid gland is ideal for in vitro organ development because this gland is relatively simple, is transplantable, and is commonly affected by a surgical complication rather than an autoimmune disease. We have investigated thymus as a source of autologous endoderm and parathyroid-like precursor cells. Human thymus cells were treated with a differentiation protocol we developed with human embryonic stem cells (The Bingham Protocol) that utilizes timed exposures to Activin A and soluble Sonic hedgehog (Shh). We incrementally changed the protocol to optimize the differentiation of the thymus cells into parathyroid-like cells. The final protocol used 50 ng/mL Activin A and 100 ng/mL Shh over 13 weeks. The differentiated cells expressed the parathyroid markers parathyroid hormone (PTH), calcium sensing receptor, chemokine receptor type-4 (CXCR4), and chorian-specific transcription factor (GCM2) as measured by reverse transcription-polymerase chain reaction and PTH enzyme-linked immunosorbent assay. Cultured thymus cells without Activin A or Shh exposure did not secrete PTH nor express similar markers. The differentiated cells released PTH, which was suppressed in response to increased calcium concentration. The chemically differentiated cells did not form tumors in immune-compromised mice. Our protocol recreated cells with markers of parathyroid tissue that responded as parathyroid cells to physiologic stimuli. This approach is a further step toward a strategy to restore parathyroid function using autologous cells that were directed to differentiate by nongenetic in vitro manipulation.

Differentiation of precursors into parathyroid-like cells for treatment of hypoparathyroidism.

BACKGROUND: Hypoparathyroidism is the most frequent permanent complication of thyroid surgery. Our hypothesis is that human precursor cells in culture can be differentiated into parathyroid cells and used to reconstitute function. Human embryonic stem cells (hESCs) are a stable model to study differentiation into parathyroid-like cells. In prior work, the BG01-hESC line was stimulated to form parathyroid-like cells. This cell line is no longer available, however, and additional studies were needed to confirm and extend prior observations. METHODS: Increasing concentrations of fetal bovine serum and timed exposure to Activin A were used to differentiate H1-hESC into parathyroid-like cells. The potential benefit of Sonic hedgehog exposure on parathyroid-like cell development also was evaluated by serial alterations of culture conditions. Calcium-sensing receptor (CaSR), GCM2, and PTH expression (RT-PCR) and PTH protein secretion (ELISA) were used as markers of differentiated cells. RESULTS: We successfully modified our prior protocol to generate cells that express CaSR, GCM2, and PTH RNA from undifferentiated H1-hESC. The cells also secreted PTH. CONCLUSION: We replicated parathyroid differentiation using H1-hESC cells. Our data advance the project toward in vitro differentiation of precursor cells isolated from individual patients for autotransplantation.

Differentiation of human embryonic stem cells to a parathyroid-like phenotype.

Iatrogenic hypoparathyroidism is the most common complication of cervical endocrine surgery. Current management is limited and palliative. As the molecular steps in parathyroid development have been defined, they may be replicable in vitro, with a goal of cellular replacement therapy. Human embryonic stem cell (hESC) lines were investigated as a model for parathyroid regeneration in vitro. BG01 was selected as a model based on expression of genes of interest in embryoid bodies (EBs). Established strategies for mouse embryonic stem cell differentiation into definitive endoderm were modified and extended to maximize the expression of definitive markers of parathyroid development. The optimal approach included the use of Activin A at 100 ng/mL with BG01 cells grown on murine embryonic fibroblasts for 5 days under conditions of increasing serum concentration. After 5 days, the cells were allowed to mature further in tissue culture without murine fibroblasts but with continuous Activin A. Our strategy produced differentiated cell cultures that expressed intermediate markers of endoderm and parathyroid development (CXCR4, EYA1, Six1, and Pax1), as well as markers of committed parathyroid precursors or developed parathyroid glands (glial cell missing-2 [Gcm2], CCL21, calcium sensing receptor [CaSR], and parathyroid hormone [PTH]). We further characterized the cells by testing conditioned medium from various time points in our differentiation scheme for the presence of PTH. We found that by keeping the cells in culture 2 weeks after the withdrawal of Activin A, the cells were able to produce PTH. Further in vivo work will be needed to demonstrate proper functionality of the cells developed in this way.

Loss of Raf kinase inhibitory protein induces radioresistance in prostate cancer.

PURPOSE: External beam radiotherapy (RT) is often used in an attempt to cure localized prostate cancer (PCa), but it is only palliative against disseminated disease. Raf kinase inhibitory protein (RKIP) is a metastasis suppressor whose expression is reduced in approximately 50% of localized PCa tissues and is absent in metastases. Chemotherapeutic agents have been shown to induce tumor apoptosis through induction of RKIP expression. Our goal was to test whether RT similarly induces apoptosis through induction of RKIP expression. METHODS AND MATERIALS: The C4-2B PCa cell line was engineered to overexpress or underexpress RKIP. The engineered cells were tested for apoptosis in cell culture and tumor regression in mice after RT. RESULTS: RT induced both RKIP expression and apoptosis of PCa cells. Overexpression of RKIP sensitized PCa cells to radiation-induced apoptosis. In contrast, short-hairpin targeting of RKIP, so that RT could not induce RKIP expression, protected cells from radiation-induced apoptosis. In a murine model, knockdown of RKIP in PCa cells diminished radiation-induced apoptosis. Molecular concept mapping of genes altered on manipulation of RKIP expression revealed an inverse correlation with the concept of genes altered by RT. CONCLUSION: The data presented in this report indicate that the loss of RKIP, as seen in primary PCa tumors and metastases, confers protection against radiation-induced apoptosis. Therefore, it is conceivable that the loss of RKIP confers a growth advantage on PCa cells at distant sites, because the loss of RKIP would decrease apoptosis, favoring proliferation.

p38MAPK-activated AKT in HER-2 overexpressing human breast cancer cells acts as an EGF-independent survival signal.

BACKGROUND: HER-2 is an epidermal growth factor receptor (EGFR) family receptor tyrosine kinase that is overexpressed in about 30% of human breast cancers correlating with a poor prognosis. Previous work in our laboratory has found that HER-2 overexpression plays a role in growth factor independence, anchorage independence, motility, and invasion of naturally occurring basement membranes. We also found that AKT was activated by p38MAPK in these cells, but this activation did not play a role in invasion. Since AKT has been shown in other systems to be a survival factor, we hypothesized that HER-2 mediated activation of AKT is necessary for growth factor independence. METHODS: Human mammary epithelial cells transduced to overexpress HER-2, HER-2, PTEN, and Myr-AKT and the primary breast cancer cell lines SUM-149 and SUM-225 were used to dissect the signaling pathways leading to growth factor independence and anchorage-independent growth in HER-2 overexpressing cells. RESULTS: We found that, in the absence of EGF, p38MAPK-activated AKT is necessary for HER-2 overexpressing cells to survive and to form colonies in soft agar. We show that EGF works as a survival signal in the absence of p38MAPK-mediated activation of AKT. We also show that human mammary epithelial cells expressing a constitutively active AKT do not require EGF for growth or colony formation in soft agar. CONCLUSIONS: The data presented here indicate that AKT activation can compensate for EGF-mediated cell survival signals leading to growth factor independence and anchorage-independent growth.

Cooperative interactions of HER-2 and HPV-16 oncoproteins in the malignant transformation of human mammary epithelial cells.

To better understand the mechanisms of transformation by the oncogene HER-2, we transduced the human mammary epithelial (HME) cell line MCF-10A with HER-2 and developed a cell line that appeared to moderately overexpress HER-2. These MCF-10HER-2 cells were unable to grow in the absence of epidermal growth factor (EGF). However, coexpression of HER-2 with the HPV-16 oncoproteins E6 and E7 resulted in EGF-independent cells that expressed very high levels of constitutively activated HER-2. Interestingly, coexpression of E7 with HER-2 resulted in cells that were EGF-independent for growth but did not express HER-2 to high levels, and coexpression of E6 with HER-2 resulted in cells expressing higher levels of HER-2, which were still dependent on EGF for growth and survival. The MCF-10HER-2E7 and HER-2/E6E7 cells exhibited constitutive activation of a form of epidermal growth factor receptor (EGFR) that had a faster electrophoretic mobility than EGFR activated by exogenous growth factors. Exposure of cells with EGFR activation to ZD1839 (Iressa), at concentrations specific for EGFR, had little or no influence on proliferation of cells with amplified HER-2 but little or no EGFR. These results indicate that HER-2, E6, and E7 cooperate with endogenous EGFR to yield fully transformed cells.

The role of phosphatidylinositol 3'-kinase and its downstream signals in erbB-2-mediated transformation.

We previously demonstrated that erbB-2-overexpressing human mammary epithelial (HME) cells exhibit several transformed phenotypes including growth factor independence, anchorage-independent growth, motility, and invasiveness. Because phosphatidylinositol 3'-kinase (PI3K) is a major target of erbB-2 activation, we tested the contribution that PI3K and its downstream signaling pathways make to these phenotypes. Utilizing a constitutively active form of PI3K, p110CAAX, we show that PI3K can mediate most phenotypes observed in erbB-2-overexpressing cells. To identify pathways leading from PI3K to specific phenotypes, we expressed constitutively active AKT or PTEN in erbB-2-overexpressing cells or in HME cells. HME cells expressing constitutively active AKT were growth factor independent, anchorage independent and motile, but not invasive. PTEN expression blocked erbB-2-mediated invasion but none of the other phenotypes. Rottlerin blocked invasion induced by p110CAAX and erbB-2, suggesting that protein kinase C delta (PKC-delta) is the downstream effector of PI3K responsible for the invasive capacity of the cells. Consistent with these observations, phospho-AKT remained detectable in erbB-2 cells treated with LY294002 or expressing exogenous PTEN, but was abolished by treatment with the p38MAP kinase inhibitor SB202190. Thus, both PI3K-dependent and p38MAP kinase-dependent pathways lead to activation of AKT, and activation of PKC-delta, via PI3K, mediates invasion.

p38MAPK induces cell surface alpha4 integrin downregulation to facilitate erbB-2-mediated invasion.

We have previously shown that human breast cancer cells that overexpress erbB-2 are growth factor-independent. In order to test the contribution of erbB-2 to this and other transformed phenotypes without the genetic instability of cancer cells, erbB-2 was overexpressed in human mammary epithelial (HME) cells. ErbB-2-overexpressing HME cells exhibit several transformed phenotypes including cell surface alpha(4) integrin downregulation and invasiveness. We formulated a model for invasiveness that depends on a cell's ability to downregulate alpha(4) integrin. As small G-proteins play a role in cytoskeleton remodeling and as this is a likely route for alpha(4) integrin trafficking, we investigated the role of small G-proteins and their downstream signals in mediating alpha(4) integrin downregulation and invasiveness using Rac 1. Dominant-negative Rac 1 blocked erbB-2-mediated invasion and reversed erbB-2-mediated alpha(4) integrin downregulation. In addition, constitutively active Rac 1 induced alpha(4) integrin downregulation and invasiveness. In erbB-2-overexpressing and in constitutively active Rac 1-expressing cells, a p38MAP kinase (p38MAPK) inhibitor blocked invasiveness and reversed alpha(4) integrin downregulation. These data suggest a model in which erbB-2 signaling activates Rac 1, which, in turn, activates p38MAPK, leading to the downregulation of alpha(4) integrin. These data strengthen the model where loss of alpha(4) integrin at the cell surface, leading to reduced alpha(4) integrin binding to plasma fibronectin, plays a role in erbB-2-mediated invasiveness.