75 CHAPTER THREE RESULTS 76 3.1 PRAP1 and intestinal differentiation 3.1.1 PRAP1 is expressed in epithelial cells of the intestines The expression of PRAP1 in human intestinal tract was studied using immunohistochemistry. Paraffin embedded tissue sections of small intestine and colon were used. In the small intestine (Figure 3.11), the expression of PRAP1 was found primarily in the epithelial cells at the top of the intestinal mucosa. PRAP1 expression was not detected in the goblet cells. There is little or no detectable expression of PRAP1 in the crypt, which is the proliferative region of the intestinal mucosa. Expression of PRAP1 was also not detected in the Paneth cells that reside at the base of crypts. A group of cells were stained very strongly for PRAP1 protein. These cells have a distribution pattern similar to that of intermediate cells which have rare occurrence in the intestinal epithelium (Troughton and Trier 1969). A similar expression pattern was observed in the colon mucosa. As shown in Figure 3.12-A, there was little or no detectable expression of PRAP1 in the goblet cells or in the crypt. Intermediate-like cells with strong PRAP1 staining were also detected. Furthermore, PRAP1 expression was localized on the luminal side of the intestinal tracts (Figure 3.12-B). This correlates with the polarization of the mucosal epithelial cells. In conclusion, our results showed that PRAP1 expression was strongly associated with the differentiation of the intestinal epithelium. Differentiation of the gut can be achieved by a number of ways. We used three different models to study the relationship between PRAP1 and the differentiation of the intestinal cells. 77 Figure 3.11 PRAP1 is expressed in the epithelial cells of small intestine Representative figure showing PRAP1 immunohistochemical staining in normal human small intestine tissue (X100 magnification). Blue hemotoxylin was used as nuclear counterstain. Red arrow indicates high levels of PRAP1 in cells at the top of the villus. Black arrow points to the crypt-villus junction. 78 Figure 3.12 PRAP1 is expressed in the epithelial cells of colon Representative figures showing PRAP1 immunohistochemical staining in normal human colon. A: Black arrow indicates positive PRAP1 staining cells at the top of the villus (100X magnification). B: Red arrow indicates high levels of PRAP1 protein in cells at the top of villus. G points to globlet cell. L indicates the luminal side of the colon (400X magnification). 79 3.1.2 Induction of PRAP1 by WNT-TCF pathway inhibition The WNT-TCF signaling pathway plays a central role in controlling the switch between proliferation and differentiation in the intestinal epithelium (Pinto and Clevers 2005; Radtke and Clevers 2005; Radtke, Clevers et al. 2006). Blocking the WNT-TCF signaling pathway results in β-catenin degradation and consequently, the ablation of β-catenin-TCF dependent gene transcription that is essential for maintaining the proliferative/undifferentiated state of intestinal epithelial cells. In this study, we used a cell line, L8, from the Clever’s lab. This cell line carries a doxycycline-inducible expression plasmid encoding Nterminally truncated TCF-4 (dnTCF-4). This truncated form cannot bind β-catenin and thus blocks the formation of endogenous β-catenin/TCF complex. As shown in Figure 3.13, there was an increase in the differentiation marker, GALECTIN-4 expression at 48 hours after inhibiting the WNT-TCF signaling pathway by dnTCF-4, indicating that induction of dnTCF resulted in the differentiation of colon epithelial cells. When the same lysate was probed for PRAP1 (Figure 3.14), it was shown that an increase in GALECTIN-4 at 48 hours correlated with an increase in PRAP1. This is consistent with our hypothesis that PRAP1 expression is correlated to cellular differentiation status. 3.1.3 Induction of PRAP1 by sodium butyrate HT 29 cells were treated with 5mM sodium butyrate to induce differentiation as documented (Zhang, Wong et al. 2003). The differentiation status of the HT 29 cells was assessed by the level of a differentiation marker, alkaline phosphatase activity. PRAP1 expression was induced by sodium butyrate at 48 and 72 hours after treatment (Figure 3.15). The induction of PRAP1 correlated significantly with the alkaline phosphatase 80 Figure 3.13 Differentiation is induced by blocking TCF4 Representative western blot of GALECTIN-4 (a differentiation marker) and GAPDH (loading control) in L8 cells after treatment with doxycycline to induce the expression of dnTCF4 (N-terminal truncated TCF) for 24, 48 and 72 hr. Figure 3.14 PRAP1 is induced in differentiated colorectal cancer cells Representative western blot of PRAP1 and GAPDH (loading control) in L8 cells after treatment with doxycycline for 24, 48 and 72 hours. 81 Figure 3.15 PRAP1 expression is induced by sodium butyrate Representative western blot of PRAP1 and GAPDH (loading control) in HT 29 cells after treatment with 5mM sodium butyrate for 24, 48 and 72 hours. r = 0.96* Figure 3.16 PRAP1 expression is correlated with differentiation A: Representative figure showing the alkaline phosphatase activities (a differentiation marker) in HT 29 cells after treatment with 5mM sodium butyrate for 24, 48 and 72 hours. B: Representative figure showing the level of PRAP1 protein expression calculated and normalized with GAPDH using densitometer in HT 29 cells after treatment with 5mM sodium butyrate for 24, 48 and 72 hours. Correlation index was calculated using GraphPad software. * p[...]... secreted into the lumen We hypothesized that PRAP1 may play a role in protecting the intestinal epithelium from invading microorganisms To verify this hypothesis, we first examined whether PRAP1 protein binds to bacteria We expressed the recombinant HisPRAP1 protein in BL 21 and purified it A high purity of HisPRAP1 was obtained as validated by the SDS-PAGE gel visualized with Coomassie Blue (Figure 3. 26)... Blue (Figure 3. 26) The binding of HisPRAP1 to E coli was 94 Figure 3. 26 Purity of HisPRAP1 protein Representative SDS-PAGE gel picture of recombinant His-tagged PRAP1 (HisPRAP1) purified by FPLC and bovine serum albumin (BSA) stained and visualized with Coomassie blue Figure 3. 27 HisPRAP1 binds to E.coli Representative figure showing absorbance reading of HisPRAP1 or BSA binding to the surface of bacteria... HisPRAP1 protein but not the BSA protein was detected on the surface of bacteria, demonstrating the binding of PRAP1 to bacteria (Figure 3. 27) Another assay was used to further verify the binding of PRAP1 to bacteria This assay involves the mixing of Alexa Fluor 568 dye labeled HisPRAP1 and BSA with fixed bacteria and incubation in the dark for 3 hours on a shaker at room temperature After washing off... differentiated epithelial cells 3. 4 .1 PRAP1 binds bacteria Since PRAP1 was expressed in the intermediate-like epithelial cells which had been shown to express antibacterial peptides, we examined whether PRAP1 protein may also play a similar functional role as that of antibacterial peptides in the gastrointestinal tract PRAP1 being a secretory protein and expressed on the luminal side of the differentiated... pcDNA-PRAP1 twice at 48 hours interval, and overexpression of PRAP1 was validated by western blot (Figure 3. 24) Overexpression of PRAP1 also did not result in any alteration in the level of GALECTIN-4 expression in LS 17 4T cells Collectively, these results demonstrate that PRAP1 protein alone is not sufficient to induce differentiation in the intestinal epithelial cells 3. 3.2 Effect of PRAP1 knockdown on cellular...85 3. 2.2 Transcriptional regulation of PRAP1 3. 2.2 .1 Promoter characterization of PRAP1 In order to study whether the regulation of PRAP1 is at the transcriptional level, various lengths of PRAP1 sequence upstream of its transcription start site were amplified from Bac clone (RP 11- 122K 13) as shown in Figure 3 .19 Each individual fragment was cloned into a firefly luciferase vector without promoter and. .. their invasion and to facilitate the clearance by professional phagocytes reside in the intestinal epithelium 3. 4.2 Bactericidal activity of PRAP1 To examine the role of PRAP1 in the innate immunity of the gastrointestinal tract, we studied its antibacterial property The pH-dependent antibacterial activity nature of PRAP1 was examined using colony forming units (CFU) assay Briefly, HisPRAP1 was incubated... differentiation in L8 cells Representative RT-PCR gel picture of prap1 and gapdh (A) and western blot of GALECTIN-4 and GAPDH (B) in L8 cells Cells were treated with or without doxycycline for 24 hours and the induced PRAP1 expression was repressed by transfecting PRAP1-specific siRNAs or control siRNA for 72 hours in the presence or absence of doxycycline Lane 1: untreated control; 2: with doxycycline; 3: control... PRAP1 overexpression on cellular differentiation To study the role of PRAP1 in cellular differentiation process, we overexpressed PRAP1 in both HT 29 cell line and LS 17 4T cell line, which is the parental cell line of L8 cells The effect of PRAP1 on cellular differentiation was analyzed by the differentiation markers, alkaline phosphatase activity in HT 29 cells, and GALECTIN-4 expression level in LS 17 4T... activities of PRAP1 were examined However, our results showed that there was no increase in the transcription activity of the core promoter, while the activity of the longest construct was increased only slightly (Figure 3. 21- B) These results suggest that the increase in PRAP1 mRNA level by cellular differentiation may not due to an increase in transcription 3. 2 .3 PRAP1 mRNA was stabilized in cellular differentiation . 76 3 .1 PRAP1 and intestinal differentiation 3 .1. 1 PRAP1 is expressed in epithelial cells of the intestines The expression of PRAP1 in human intestinal tract was studied using immunohistochemistry differentiation of the intestinal cells. 77 Figure 3 .11 PRAP1 is expressed in the epithelial cells of small intestine Representative figure showing PRAP1 immunohistochemical staining in normal. epithelium from invading microorganisms. To verify this hypothesis, we first examined whether PRAP1 protein binds to bacteria. We expressed the recombinant HisPRAP1 protein in BL 21 and purified