Proline rich acidic protein 1 in life and death 3

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

CHAPTER THREE

RESULTS

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

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

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)

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 N-terminally 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

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

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

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<0.05

r = 0.96*

activity (R=0.96, Figure 3.16) Together, our results lend support to our observation that PRAP1 is associated with differentiation

3.1.4 Induction of PRAP1 by glucose deprivation

HT 29 cells can be induced to differentiate by the removal of glucose (Zweibaum, Pinto et al 1985; Liu, Huang et al 2006) Consistent with the reported results, HT 29 cells with the ability to grow without glucose showed an increase of more than 2-fold in the alkaline phosphatase activities as compared to the untreated control cells (Figure 3.17-A) The expression of PRAP1 was increased by 2.5-fold in these differentiated cells compared to the control cells without glucose deprivation (Figure 3.17-B) Together, our results support our conclusion that PRAP1 is strongly associated with differentiation

3.2 Regulation of PRAP1 expression by differentiation

3.2.1 Induction of PRAP1 expression at mRNA level

To further study the regulation of PRAP1 by differentiation, we examined

the induction of PRAP1 expression at mRNA level using quantitative RT-PCR

As shown in Figure 3.18-A, PRAP1 mRNA expression level was increased at 48

hours after the induction of dnTCF, and further increased to 4.5-fold at 72 hours

Similar results were observed in the sodium butyrate model PRAP1 mRNA

expression level was increased at 48 hours after sodium butyrate treatment, and further increased to 5-fold at 72 hours (Figure 3.18-B) These data indicate that induction of PRAP1 occurred by an increase of PRAP1 mRNA

Figure 3.17 PRAP1 expression is induced by glucose deprivation

A: Representative figure showing the alkaline phosphatase activity (a differentiation marker) in HT 29 cells after subjecting to glucose deprivation B: Representative western blot of RPAP1 and GAPDH (loading control) in HT 29 after glucose deprivation treatment (Glu-)

Figure 3.18 PRAP1 expression is induced at mRNA level by differentiation

Representative figures showing quantitative RT-PCR of PRAP1 mRNA expression level normalized with GAPDH (housekeeping gene) in L8 cells after treatment with doxycycline for 24, 48 and 72 hours (A); and in HT 29 cells after treatment with 5mM sodium butyrate for 24, 48 and 72 hours (B)

Figure 3.19 Cloning of PRAP1 promoter

Representative agarose gel pictures of PCR cloning products of various lengths of DNA sequence upstream of PRAP1 transcription start site amplified from BAC clone The size and position of each DNA fragments was indicated in the agarose gel pictures

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 (RP11-122K13) as shown in Figure 3.19 Each individual fragment was cloned into a firefly luciferase vector without promoter and enhancer (pGL3-Basic) as shown in Figure 3.20-A These reporter constructs

containing different lengths of the PRAP1 promoter showed a varied degree of

promoter activity, suggesting the presence of regulatory elements Majority of the

PRAP1 promoter constructs showed a promoter activity of more than 15-fold as

compared with that of the empty vector The lowest promoter activity was

observed in the promoter construct with 3000 base pairs upstream of the PRAP1

transcription start site The activity of the shortest fragment, pGL(-203/0) was similar as that of larger fragments, suggesting the presence of a core promoter (Figure 3.20-A)

To further confirm the location of the PRAP1 core promoter, a deletion

construct pGL(-461/-203) was generated by deleting the 203 base pairs upstream

of the transcription start site Consistent with our observation, the activity of the deletion construct pGL(-461/-203) decreased to that of basic vector (Figure 3.20-

B) These results indicate that the core promoter of PRAP1 was located within 203 base pairs upstream of the PRAP1 transcription start site

Figure 3.20 PRAP1 promoter activities in L8 cells

A: Left panel: Illustration representing various portions of the prap1 5’ flanking region of the prap1 gene subcloned upstream of the firefly luciferase gene (pGL3- Basic) The number in the box indicates the size of each prap1 fragment with

respect to the transcription start site (+1)

Right panel: Representative figure showing the fold induction of prap1 promoter

activity of each fragment in L8 cells For each transfection, the firefly luciferase activity was normalized with the Renilla reniformis luciferase activity by the cotransfected pRL-TK The relative activity of each construct is expressed as a ratio to the activity of the pGL-Basic Bar, mean of three replicates Bar, SE B: Left panel: Illustration showing the deletion construct pGL(-461/-203) generated by deleting the 203 base pairs upstream of the transcription start site from the pGL(-461/0) construct

Right panel: Representative figure showing the fold induction of prap1 promoter

activity of each construct in L8 cells The promoter activity was measured as described in A Column, mean of three replicates; Bar, SE

3.2.2.2 PRAP1 expression was not regulated at transcriptional level

To study the regulation of PRAP1 at transcriptional level, two reporter constructs of PRAP1 promoter, the longest construct pGL(-3900/0) and the core promoter pGL(-203/0) were used These constructs were transfected into L8 cells

At 24 hours post-transfection, doxycycline was added to induce differentiation

An increase in PRAP1 mRNA by differentiation was confirmed by RT-PCR

(Figure 3.21-A) At the same time, the promoter 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

Since PRAP1 expression is not regulated at the transcriptional level by differentiation, we examined whether it was due to an increase in the stability of

PRAP1 mRNA L8 cells were treated with doxycycline for 72 hours and total

mRNA synthesis was inhibited by treating the cells with actinomycin (4µg/ml) for

0, 2 and 4 hours before harvesting Our results showed that the stability of PRAP1

mRNA was dramatically enhanced after induction of dnTCF-4 (Figure 3.22) At 2

hours time-point, PRAP1 mRNA level in the more differentiated L8 cells showed

a slower rate of reduction as compared to the control undifferentiated L8 cells

(10% vs 25%) At 4 hours, the level of PRAP1 mRNA in the undifferentiated cells was further reduced by 70%, whereas the PRAP1 mRNA level in the

results indicate that the increase in PRAP1 mRNA levels following differentiation

occurs at the post-transcriptional level through stabilization of mRNA

Figure 3.21 PRAP1 is not regulated at transcriptional level by differentiation

A: Representative RT-PCR gel picture of prap1 and gapdh in L8 cells after

treatment with (+) or without (-) doxycycline for 72 hours

B: Representative figure showing the luciferase activity of prap1 promoter

constructs pGL(-3900/0) and pGL(-203/0) in L8 cells after treatment with doxycycline for 72 hours Luciferase activity was calculated as in Figure 3.20 Column, mean of three replicates; Bar, SE

Figure 3.22 Stability of PRAP1 mRNA is increased by differentiation

Representative figure showing the percentage of PRAP1 mRNA expression level

in L8 cells Cells were treated with (+) or without (-) doxycycline for four days and 4 µg/ml of actionomycin was added for 0, 2 and 4 hours to stop endogenous

mRNA synthesis Relative expression level of PRAP1 was measured and normalized with GAPDH using quantitative RT-PCR Level of expression at 0

hour was used as a reference point Level of PRAP1 expression was expressed as

a percentage of the reference point

3.3 Effect of PRAP1 on differentiation

3.3.1 Effect of 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 174T 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 174T cells Our results showed that PRAP1 was overexpressed in HT 29 cells by transient transfection of PRAP1 gene construct plasmid (pcDNA-PRAP1) for 72 hours (Figure 3.23-A) However, overexpression of PRAP did not result in any changes in the activities of alkaline phosphatase in HT 29 cells (Figure 3.23-B) Similar results were also observed in

LS 174T cells LS 174T cells were transiently transfected with 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 174T 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 differentiation

The role of PRAP1 in differentiation was further explored by examining the effect of inhibiting the induction of PRAP1 on the differentiation status To achieve this, we used siRNAs to knockdown the induction of PRAP1 mRNA during differentiation Briefly, LS 8 cells were first treated with doxycycline for

24 hours to induce PRAP1 expression Two siRNAs specific for PRAP1 were transiently transfected to repress this