Proline rich acidic protein 1 in life and death r6

Figure 3.74 PRAP1 expression is increased in apoptotic cells A: Representative figure showing the relative expression level of PRAP1 mRNA level in the adherent control and floating Apop

to overexpress PRAP1 before subjecting them to 5-FU treatment Lysates were harvested and immunoprecipitation were carried out using antibody against Hsp

70 The immunoprecipitated proteins were then analyzed by western blotting As shown in Figure 3.72, PRAP1 and Hsp 70 coprecipitated PRAP1 was detected in Hsp 70 precipitates These data demonstrated that Hsp 70 interacted with PRAP1 and were present in the same complex upon 5-FU treatment

3.12 Role of PRAP1 in apoptotic cells

In our study, we observed that PRAP1 was highly expressed in both differentiated cells as well as stress-induced apoptotic cells In addition, the protein PRAP1 was found to bind to the surface of bacteria cells We hypothesized that PRAP1 may also bind to the surface of apoptotic cells and may

play a role in the clearance of apoptotic cells

3.12.1 Induction of PRAP1 expression in apoptotic cells

To further validate the expression of PRAP1 in apoptotic cells, we used

HT 29 cells which have high basal levels of PRAP1 protein expression Briefly,

HT 29 cells were treated with 5mM sodium butyrate for 48 hours to induce apoptosis Cell culture medium containing dead cells that were detached from the culture flask were harvested The live cells that remained attached to the culture flask were harvested by trypsinization Cells were stained with propidium iodide and subjected to flow cytometry for analysis of DNA content Our results showed that the population of the attached cells in sub-G1 was comparable to that of the untreated control (Figure 3.72) On the other hand, the floating cells showed a significant increase in the sub-G1 population This indicated that the floating cells were composed of mainly dead cells

Figure 3.72 Dying cells in floating population

Representative histogram showing HT 29 cells untreated (control) or treated with 5mM sodium butyrate for 48 hours Floating cells (Floating) from the treated sample were harvested separately from the adherent cells (Adherent) All cells were fixed and stained with propidium iodide and DNA content was analyzed by flow cytometry Box indicates cells at Sub-G1 (dying cell population)

To further validate the apoptotic nature of these floating cells, we employed the annexin V-FITC staining assay Annexin V is a phospholipid binding protein that has a high binding affinity for phosphatidylserine (PS) that translocates from inner to outer leaflet of plasma membrane during early apoptosis As shown in Figure 3.73, there was a shift of the fluorescent peak to the right in the floating cells as compared to that of the adherent cells There was minimal annexin V staining in the adherent cells, as indicated by a lack of shift in the fluorescent peak when compared to that of the untreated control The population of cells with positive annexin V staining was calculated by setting the gating at R1 as shown in Figure 3.73 In the floating cells, 83% of cells were positive for annexin V expression, while only 14% of adherent cells and 12% of the untreated control were positive These results demonstrated that the majority

of the floating cells were apoptotic cells, suggesting that we have successfully obtained two distinct populations of cells, the live cells (attached cells) and the apoptotic cells (floating cells)

The expression of PRAP1 in these two populations was analyzed Our

results showed that PRAP1 mRNA was highly induced in the apoptotic cells as

compared to the adherent cells (Figure 3.74-A) Consistent with an increase at the mRNA level, PRAP1 protein was also increased in the apoptotic cells (Figure 3.74-B) These results showed that PRAP1 expression was highly induced in

apoptotic cells compared to the live cells

3.12.2 PRAP1 binds to the surface of apoptotic cells

As PRAP1 is a secreted protein, we investigated whether PRAP1 binds to the surface of the apoptotic cells Apoptotic cells were harvested and either labeled with anti-PRAP1 antibody directly or fixed with 4% paraformaldehyde,

Figure 3.73 Apoptotic cells in floating population

Representative histogram showing annexin-V staining in HT 29 cells after treatment with 5mM sodium butyrate for 48 hours or untreated (blue) Floating cells (green) and attached cells (red) from the treated sample were harvested separately All cells were fixed and stained with annexin-V conjugated to FITC and analyzed by flow cytometry R1 gates the population of cells with positive annexin-V staining

Figure 3.74 PRAP1 expression is increased in apoptotic cells

A: Representative figure showing the relative expression level of PRAP1 mRNA level in the adherent (control) and floating (Apoptotic cells) of HT 29 cells after treatment with 5mM sodium butyrate for 48 hours

B: Representative western blot of PRAP1 and GAPDH (loading control) of the adherent (control) and floating (Apoptotic) population of HT 29 cells after treatment with 5mM sodium butyrate for 48 hours

proteins were detected on the surface of both unfixed and fixed apoptotic cells (Figure 3.75) The staining was not homogenous, with some cells showing weak

followed by immunofluorescence staining with PRAP1 antibody Our results showed that PRAP1 observation was confirmed using flow cytometry As shown

in Figure 3.76, there were two populations of cells as indicated by two peaks (shaded in red) Those cells with higher PRAP1 (black arrow) on the surface were detected by a right shift as compared to the IgG control There was a population with no or little detection of PRAP1 (red arrow) This observation was further confirmed to be not cell line specific We repeated the experiment in another cell line, HCT 116 Briefly, HCT 116 was treated with 3mM sodium butyrate and apoptotic cells were harvested after 48 hours for immunofluorescence staining Consistent with our previous finding, PRAP1 was detected on the surface of apoptotic cells (Figure 3.77) Together, our results showed that PRAP1 was induced in the apoptotic cells and bound onto the surface of the apoptotic cell

The binding of PRAP1 on the surface of apoptotic cells were confirmed using two additional methods: direct immunofluorescence and transmission electron microscopy In the direct immunofluorescence assay, apoptotic cells were fixed and incubated with recombinant HisPRAP1 protein conjugated to Alexa fluor dye Our results showed that exogenous PRAP1 was detected on the surface

of apoptotic cells (Figure 3.78) Both free dye and BSA conjugated to Alex fluor dye were not detected on the surface of apoptotic cells In the transmission electron microscopy (TEM) assay, apoptotic cells were fixed, stained with PRAP1-specific antibody and detected using secondary antibody conjugated to gold particles Cells were processed for TEM and pictures were taken As shown

in Figure 3.79, there were gold particles, as indicated by the red arrows, detected

on the

Figure 3.75 Immunofluorescence images showing the binding of PRAP1 to the surface of apoptotic cells

Representative immunofluorescence images of PRAP1 binding to the floating population of HT 29 cells after treatment with 5mM sodium butyrate for 48 hr Floating cells were either unfixed or fixed in 4% paraformaldehyde and immunofluorescence was performed by incubating with anti-PRAP1 antibody (1:200) for 1 hour, followed by secondary anti-rabbit antibody conjugated to Alexa Fluor 488 dye (Green)

Figure 3.76 Histogram showing PRAP1 binds to the surface of apoptotic cells

Representative histogram of PRAP1 (shaded in red) and rabbit IgG isotype (unshaded) staining in floating population of HT 29 cells after treatment with 5mM sodium butyrate for 48 hours Cells were either fixed or unfixed in 4% paraformaldehyde Red arrow indicates the fluorescence peak with unstained population Black arrow indicates fluorescence peak with positive staining

Figure 3.77 PRAP1 binds on the surface of HCT 116 apoptotic cells

Representative immunofluorescence images of PRAP1 binding to apoptotic HCT

116 cells after treatment with 5mM sodium butyrate for 48 hours

Figure 3.78 Alexa fluor labeled HisPRAP1 binds directly to the surface of apoptotic cells

Representative immunofluorescence images of floating population of HT 29 cells obtained after treatment with 5mM sodium butyrate for 48 hours incubated with alexa fluor labeled HisPRAP1 (PRAP1) or free alexa fluor dye Images were taken using Confocal microscope

Figure 3.79 Transmission electron microscopy of PRAP1 on apoptotic cells

Representative transmission electron microscopy (TEM) images of apoptotic HT

29 cells stained with PRAP1 antibody PRAP1 protein is indicated by red arrows and apoptotic bleb is indicated by black arrow

outer surface of the membrane One gold particle was detected on the surface of a bleb (as indicated by a black arrow) These data showed for the first time that PRAP1 binds on the outer surface of cell membrane of apoptotic cells

3.12.3 PRAP1 enhanced the phagocytosis of beads

As we have shown previously that PRAP1 binds to the surface of bacteria,

we investigated whether the presence of PRAP1 on the surface of apoptotic cells would affect their clearance by phagocytosis To achieve that, we used FluoSpheres polystyrene with yellow-green fluorescence Briefly, these microspheres were either coated with HisPRAP1, BSA or uncoated These coated beads were then incubated with a monocytic cell line, U937 Microspheres that were not ingested by U937 cells were quenched Population of cells with ingested beads was measured by flow cytometry Figure 3.80-A showed the representative pictures of scatter-histogram plot analysis of phagocytosis of beads Cells with ingested beads are gated (as indicated by the circle) and percentage of cells with ingested beads was calculated and summarized in Figure 3.80-B Coating of PRAP1 onto the surface of microspheres significantly enhanced their uptake by U937 At as early as 15 min, coating of PRAP1 resulted in more than 3-fold increase in the ingestion of the microspheres as compared to the BSA coated microspheres control (16% vs 4%) After 1 hour, microspheres coated with PRAP1 were ingested by about 50% of the cells while only 13% of cells had ingested the BSA coated microspheres

To mimic the in vivo system, we differentiated the monocytic cell line,

U937 into a macrophage lineage by treating it with dibutyryl cyclic adenosine monophosphate (dcAMP) As shown in Figure 3.81, these macrophages were

Figure 3.80 Flow cytometry analysis of phagocytosis of beads by U937

Representative scatter plot of phagocytosis of beads (A) Fluorescent beads uncoated or coated with BSA or HisPRAP1 were incubated with U937 cells and phagocytosis were analyzed with flow cytometry U937 cells with ingested fluorescent beads were positive with fluorescent and gated (circle) and expressed

as a percentage of total cells (B) Number on top of column indicates the mean Columns, mean of 3 replicates; Bars, SE

Figure 3.81 HisPRAP1 enhances phagocytosis of beads by macrophages

Representative figure showing the percentage of macrophages with ingestion of beads after incubating with beads alone or beads coated with BSA or HisPRAP1 (Prap) for 20, 40, 60 and 90 minutes Number on top of each column indicates mean Columns, mean of three replicates; Bars, SE

more phagocytic than their undifferentiated parental cells At as early as 20 min, these macrophages ingested more than 2-fold of uncoated beads as compared to undifferentiated U937 cells (11% vs 5%) Consistent with our finding, PRAP1 coated microspheres significantly enhanced the phagocytosis process by more than 3-fold as compared to BSA coated microspheres (35% vs 9%) The results suggest that cells undergoing apoptosis increase the secretion of PRAP1 which binds on the surface of the apoptotic cells, which may enhance the clearance of these cells by increasing the phagocytosis of apoptotic cells by macrophages

3.13 Role of PRAP1 in a disease model, SLE

3.13.1 Detection of PRAP1 autoantigen

As there is growing evidence for an impairment of clearing apoptotic cells

in mouse models of SLE (Taylor, Carugati et al 2000; Licht, Dieker et al 2004) and in humans (Herrmann, Voll et al 1998; Baumann, Kolowos et al 2002), we hypothesized that PRAP1 may play a role in the clearance of apoptotic cells in this disease Since SLE is characterized by the presence of autoantigens and autoantibodies, we examined the presence of PRAP1 protein and its autoantibody

in the serum of SLE patients As shown in Figure 3.82-A, PRAP1 protein was detected in the serum of both normal and SLE patients The concentration of PRAP1 in SLE patients was heterogeneously distributed There were 28% of SLE patients with higher PRAP1 protein (mean ± 2SD) detected in the serum as compared to the normal (18 out of 65, Figure 3.82-B) There were 13% of SLE patients with lower PRAP1 level and two of the patients had ten-fold less PRAP1 compared to the normal individuals

Figure 3.82 PRAP1 protein was detected in the serum of both normal and SLE patients

A: Scatter plot showing the distribution of PRAP1 protein level in serum Level of PRAP1 protein in the serum of normal individuals and SLE patients was assayed

by ELISA HisPRAP1 was used as a standard to calculate the amount of PRAP1 protein present in the serum

B: Summary of the ELISA data showing two populations of SLE patients expressing lower or higher PRAP1 than the normal individual Column, mean;

Bar, SE * p<0.001

3.13.2 Detection of PRAP1 autoanitbodies

To detect PRAP1 autoantibodies, HisPRAP1 was used as antigen in an ELISA assay Majority of SLE patients had similar level of PRAP1 autoantibodies

as that of the normal individuals (Figure 3.83-A) The mean of the PRAP1 autoantibodies in SLE patients was slightly higher than that of the normal and not significant (Figure 3.83-B) There were 11% of SLE patients (9 out of 85) showing highly elevated PRAP1 autoantibodies (mean + 2SD) The specificity of these autoantibodies was verified by western blot Briefly, HisPRAP1 protein was loaded unto SDS-PAGE gel and transferred unto nitrocellulose membrane and the membrane was probed with serum from three SLE patients with either high or low autoantibody titer Consistent with our ELISA result, only serums from patients with high titer of PRAP1 autoantibodies were able to detect HisPRAP1 on the nitrocellulose membrane (Figure 3.84) Moreover, the band was specific as compared to that of the positive control These results confirmed the titer of the PRAP1 autoantibody as measured by ELISA, and the autoantibodies detected were specific against PRAP1 These results showed that PRAP1 autoantibodies are present in the serum of both normal and SLE patients

3.13.3 PRAP1 and proinflammatory cytokines

As PRAP1 autoantigens and autoantibodies were detected in SLE patients,

we investigated whether PRAP1 plays a role in immune cells interaction To achieve this, we used ELISA to measure some of the common proinflammatory cytokines (TNFα, IL-6 and IL-1β) that are associated with acute inflammation Briefly, monocytes were stimulated by activated T-cells to produce cytokines In this study, we activated Jurkat

Figure 3.83 PRAP1 autoantibody was detected in serum

A: Scatter plot showing the distribution of PRAP1 autoantibodies level in serum Level of PRAP1 autoantibodies in the serum of normal individuals and SLE patients was assayed by ELISA OD50 was calculated for each individual samples using the GraphPad software

B: Summary of the OD50 of PRAP1 autoantibody obtained for both the normal

and SLE patients Column, mean; Bar, SE

Figure 3.84 Verification of PRAP1 autoantibodies in the serum

Representative western blot of HisPRAP1 detected using the PRAP1 autoantibodies in the serum of SLE patients Lane 1: polyclonal anti-PRAP1 antibody; Lane 2-4: SLE patients with high titer of PRAP1 autoantibodies; Lane

5-7: SLE patients with low titer of PRAP1 autoantibodies

cells with PHA/PMA, fixed them and co-cultured with monocytes (U937) in the presence of various concentration of HisPRAP1 Supernatant were harvested after

48 hours and cytokines were assayed by ELISA As shown in Figure 3.85, U937 cells were not able to produce cytokines by themselves or when co-cultured with unstimulated Jurkat cells Activated Jurkat cells were fixed and shown not to be producing any cytokines Upon co-culture with activated Jurkat cells, U937 produced high amount of proinflammatory cytokines However, addition of HisPRAP1 at low concentrations ranging from 1ng/ml to 200ng/ml did not result

in any significant changes in the production of the proinflammatory cytokines This observation was similar with higher concentration of HisPRAP1 ranging from 500ng/ml to 10,000ng/ml (Figure 3.86) These results suggest that PRAP1 does not play a role in regulating immune cells interaction

3.13.4 PRAP1 expression in PBMC

To study whether PRAP1 expression is differentially regulated by lymphocytes from normal and SLE patients, we isolated PBMC from the blood of

15 normal individuals and 40 SLE patients RNAs were extracted and the level of

PRAP1 expression in the PBMC of both normal and SLE patients were measured

by quantitative real-time RT-PCR There were about 90% of SLE patients (36 out

of 40) with lower expression of PRAP1 as compared to that of the normal individuals (Figure 3.87-A) The expression of PRAP1 was reduced significantly

by more than 2-fold when compared to that of the normal individuals (Figure 3.87-B) This observation was consistent with our hypothesis that the absence of PRAP1 may play a role in the impairment of clearance of apoptotic cells as observed in SLE

TNF-a production

0 1000

200 Tc/U/P 100 Tc/U/P 50 Tc/U/P 10 Tc/U/P 1

Ts Ts/U Ts/U/P 200 Ts/U/P 100 Ts/U/P 50 Ts/U/P 10 Ts/U/P 1

Ts Ts/U Ts/U/P 200 Ts/U/P 100 Ts/U/P 50 Ts/U/P 10 Ts/U/P 1

M U U/P

200U/P

100U/P

50U/P

10U/P

1 Tc T

Tc/U/P 00

Tc/U/P 00

Tc/U/P 0

Tc/U/P 0

Tc/U/P Ts T

Ts/U/P 00

Ts/U/P 00

Ts/U/P 00

Ts/U/P 0

Ts/U/P

TNFa production

0 1000 2000 3000 4000 5000 6000 7000

10000 U/P 5000 U/P 1000 U/P 500

Tc Tc/U Tc/U 10000 Tc/U/P 5000 Tc/U/P 1000 Tc/U/P 500

Ts Ts/U Ts/U/P 10000 Ts/U/P 5000 Ts/U/P 1000

10000 U/P 5000 U/P 1000 U/P 500

Tc Tc/U Tc/U 10000 Tc/U/P 5000 Tc/U/P 1000 Tc/U/P 500

Ts Ts/U Ts/U/P 10000 Ts/U/P 5000 Ts/U/P 1000 Ts/U/P 500

10000 U/P 5000 U/P 1000 U/P 500

Tc Tc/U Tc/U 10000 Tc/U/P 5000 Tc/U/P 1000 Tc/U/P 500

Ts Ts/U Ts/U/P 10000 Ts/U/P 5000 Ts/U/P 1000 Ts/U/P 500

by ELISA M, media alone; U, U937 cells; Tc, Unstimulated Jurkat cells; Ts, activated Jurkat cells; Tc/U, Tc co-cultured with U; Ts/U, Ts co-cultured with U; Tc/U/P, Tc/U in the presence of HisPRAP1; Ts/U/P, Ts/U cultured in the presence

of HisPRAP1 Amount of each proinflammatory cytokines were calculated based

on their respective standard curve Column, mean of three replicates; Bar, SE

Figure 3.87 PRAP1 mRNA expression is reduced in SLE patients

A: Representative figure showing the relative expression level of PRAP1 mRNA

in the PBMC isolated from the blood of normal (n=15) and SLE patients (n=40) PRAP1 level was assayed by real-time RT-PCR Expression of PRAP1 was normalized with GAPDH Column, mean of 3 replicates; Bar, SE

B: Summary of real-time RT-PCR data showing PRAP1 in SLE patients were

reduced by 2-fold Column, mean; Bar, SE * p<0.05

3.14 Regulation of PRAP1 expression in lymphocytes

3.14.1 PRAP1 is induced by PHA/PMA

To investigate the regulation of PRAP1 in lymphocytes by various cell death stimuli, we used PHA/PMA, UV and cytotoxic drugs to induce apoptosis

We first examined the effect of PHA/PMA on PRAP1 expression PHA and PMA

are potent mitogens for immune cells Both of them are able to induce apoptosis in Jurkat cells (Abbady, Bronner et al 2003) Briefly, Jurkat cells were activated with PHA (5μg/ml) and PMA (5μM) Cells were harvested at 2, 4, 6, 8, 10, 12 and

24 time points As shown in Figure 3.88, PRAP1 mRNA expression was induced

at early time points It was induced at as early as 2 hours after activation, and

maintained for the next 6 hours of treatment At 10 hours after activation, PRAP1

level was reduced back to its basal level for the next 14 hours This result indicated that apoptosis inducing agent, PHA/PMA can induce the expression of

PRAP1

3.14.2 PRAP1 is induced by UV

Four different lymphocytic cell lines, HL-60, U937, Raji and THP-1 were

used to study the regulation of PRAP1 by UV Briefly, cells were exposed to

various dosage of UV and recovered for 4 hours in an incubator Cells were then

harvested and the level of PRAP1 expression was analyzed using real-time PCR PRAP1 expression was induced in a dose-dependent manner in all the four cell lines (Figure 3.89) The expression of PRAP1 was induced at about 3-fold in

RT-HL-60, U937 and Raji cells, and 15-fold in THP-1 cells at the highest UV dose

used These data demonstrated that PRAP1 was increased in lymphocytes upon

exposure to UV

Figure 3.88 PRAP1 expression is induced by PHA/PMA

Representative RT-PCR gel picture of prap1 and gapdh in Jurkat cells after

treatment with PHA/PMA for the indicated time points

Figure 3.89 PRAP1 expression is induced by UV

Representative figure showing the fold induction of PRAP1 mRNA in four lymphocytes cell lines (U937, Raji, THP-1 and HL-60) after exposure to UV at the stated dosage (J/m2) PRAP1 expression level was assayed by probe-based real-time RT-PCR and normalized with GAPDH Column, mean of 3 replicates; Bar, SE

3.14.3 PRAP1 is induced by cytotoxic drugs

Four different lymphocytic cell lines which made up of monocytic (U937, HL-60 and THP-1) and T-cell (Jurkat) lines were treated with camptothecin (CPT), etoposide (Eto) and 5-Fluorouracil (5-FU) for 48 hours to induce cell

death 5-FU was the most potent in inducing PRAP1 expression in U937, THP-1 and Jurkat (Figure 3.90) We failed to detect any significant induction of PRAP1

by 5-FU in HL-60 cells PRAP1 expression was also induced by CPT and Eto in

all the four cell lines with the exception of THP-1 cells upon Eto treatment These

results indicate that regulation of PRAP1 by genotoxic agents was both cell line-

and drug-specific

3.14.4 Regulation of PRAP1 expression in PBMC by cytotoxic drugs

In order to study the regulation of PRAP1 expression in normal and SLE

patients, we isolated PBMC from buffy coat of normal individuals and blood of SLE patients As shown in Figure 3.91, upon induction of cell death by CPT, Eto

and 5-FU, PRAP1 expression was highly induced in PBMC from normal

individuals This increase was dose-dependent for 5-FU and Eto but was reversed with regard to CPT (Figure 3.91) At as low as 2 µM of 5-FU, 0.1 µM of CPT and

0.8 µM of Eto, expression of PRAP1 was upregulated in the PBMCs However, in PBMC from SLE patients, expression of PRAP1 failed to be induced by all these

three different cytotoxic drugs, even at the highest dosage (Figure 3.92) To examine closely the regulation of PRAP1 expression in these SLE patients, we used a quantitative RT-PCR assay Our results showed that only one out of the five SLE patients (20% vs 80%) demonstrated a 1.5-fold and 2-fold increase when

Figure 3.90 PRAP1 expression is induced by cytotoxic drugs in lymphocytes

Representative RT-PCR gel picture of prap1 and gapdh in lymphocytes cell lines (Jurkat, HL-60 U937 and THP-1) after exposure to cytotoxic drugs (10 µM Camptothecin, CPT; 80 µM Etoposide, Eto; 200 µM 5-Fluorouracil, 5-FU) for 48

hr Total RNAs were extracted and PRAP1 expression level was assayed by PCR GAPDH was used as a housekeeping gene

RT-Figure 3.91 PRAP1 is induced in normal PBMC by cytotoxic drugs

Representative RT-PCR gel picture showing the mRNA expression level of prap1 and gapdh (housekeeping gene) in PBMCs isolated from normal individuals after

treatment with different doses of cytotoxic drugs (Camptothecin, CPT; Etoposide, Eto; 5-Fluorouracil, 5-FU) for 48 hours

Figure 3.92 PBMCs from SLE patients failed to induce PRAP1

Representative RT-PCR gel picture showing the regulation of PRAP1 mRNA by cytotoxic drugs in two representative SLE patients (SLE1 and SLE 3) PBMCs were isolated from SLE patients and untreated (C) or treated with cytotoxic drugs (10 µM Camptothecin, CPT; 80 µM Etoposide, Eto; 200 µM 5-Fluorouracil, 5-FU) for 48 hours Total RNAs were extracted and PRAP1 expression level was assayed by RT-PCR GAPDH was used as a housekeeping gene

Figure 3.93 PBMCs from SLE patients failed to induce PRAP1 mRNA

A: Representative figure showing the fold induction of PRAP1 mRNA expression level in PBMCs was isolated from five SLE patients after treatment with cytotoxic drugs (10 µM Camptothecin, CPT; 80 µM Etoposide, Eto; 200 µM 5-

Fluorouracil, 5-FU) for 48 hours Total RNAs were extracted and prap1 and

gapdh expression level was assayed by quantitative real-time RT-PCR GAPDH

was used as a housekeeping gene Level of PRAP1 mRNA induction was expressed as fold induction over that of untreated control Column, mean of duplicates; Bar, SEM

B: Summary of relative mRNA expression levels of PRAP1 quantified by time RT-PCR in PBMCs isolated from five SLE patients Column, mean of five replicates; Bar, SEM

real-A

B

treated with 5-FU and Eto respectively (Figure 3.93-A) The expression of PRAP1 was reduced in PBMCs from the SLE patients after treatment with all the three different cytotoxic agents (Figure 3.93-B) This is consistent with our hypothesis that PRAP1 expression may play a role in the clearance of apoptotic cells Failure

to induce PRAP1 expression may be one of the factors that accounts for the

impaired clearance of apoptotic cells in SLE

CHAPTER FOUR

DISCUSSION

4.1 Role of PRAP1 in differentiated epithelial cells

4.1.1 Regulation of PRAP1 by differentiation

4.1.1.1 Expression of PRAP1 in intestinal epithelium

The proline-rich acidic protein (PRAP1) gene was first identified by Kasik

et al In the article, the PRAP1 gene was found to be expressed in the mouse as a pregnant-specific uterine protein (Kasik and Rice 1997) The PRAP1 gene was

expressed in the mouse uterus from day 12 of pregnancy to day 3 after parturition The rat homologue was later isolated and characterized in our laboratory (Zhang,

Rajkumar et al 2000) The mRNA sequence of PRAP1 of rat and mouse shared 90% homology The PRAP1 gene encoded a putative protein of 152 amino acids

in rat, and 150 in mouse The predicted amino acid sequences of rat and mouse

shared 84% homology The expression of PRAP1 in rat was found not to be limited to the late pregnant uterus PRAP1 transcripts were detected in the epithelial cells of the gastrointestinal tract The human homologue of PRAP1 was

also isolated and characterized in our laboratory (Zhang, Wong et al 2003) Human PRAP1 cDNA encoded a putative protein of 151 amino acids and shared 50% homology to that of rat and mouse The NH2 terminus was highly conserved among human and rodent, and was predicted to be a signal peptide In addition, the COOH terminus comprising amino acids from position 135 to 149 was 80% conserved between human and rodent The predicted cleavage site was between amino acid 20 and 21 PRAP1 was shown to be a secreted protein (Zhang, Wong

et al 2003) The human PRAP1 gene was found to be expressed in the epithelial cells of gastrointestinal tract, kidney and liver

The PRAP1 gene was commonly expressed in the epithelial cells of the

gastrointestinal tract of both human and rodent, suggesting a conserved function

of PRAP1 in the intestine during development Interestingly, the expression of

PRAP1 gene was differentially regulated, with a proximal-distal gradient

expression pattern, with higher expression in the proximal gastrointestinal tract These suggest that PRAP1 may be involved in the differentiation of epithelial cells during development and/or in the constitutive cell renewal program that maintains the gastrointestinal epithelium

In this study, the expression of PRAP1 in the intestinal epithelium was further elucidated Our results showed that PRAP1 protein was expressed abundantly in the epithelium of both small intestine and colon PRAP1 expression was located at the top of the crypt consisting of differentiated cells but not at the base of the crypt, which is the proliferative stem cell region This strongly indicates that PRAP1 is associated with epithelial differentiation The type of cells that express PRAP1 could be either the enterocytes or the neuroendocrine cells, but not the goblet cells that secrete mucus Interestingly, a small subset of cells was highly stained with PRAP1 expression This staining pattern was detected in both small intestine and colon, with higher frequency in the small intestines The distribution pattern of these cells is similar to that of a new type of intestinal epithelial cell described by Troughton et al., called the intermediate cell These cells have rare occurrence and was also reported to express enteric α-defensin, human defensin 5 (HD-5), which plays an important role in mediating innate immunity of the gastrointestinal tract (Cunliffe, Kamal et al 2002) Co-staining for PRAP1 and human α-defensin 5 may help to address the identity of those cells

types suggests that PRAP1 may be a protein with multiple functions which may

be cell type-specific

4.1.1.2 PRAP1 expression is positively correlated with differentiation

The intestinal epithelial cells undergo apoptosis and/or extrusion into the lumen every three to five days This rapid turnover requires a continual coordinated series of events that include cell proliferation, lineage commitment, and cell differentiation throughout the postnatal life of mammalian cells (Potten and Loeffler 1990) In this study, the regulation of PRAP1 by differentiation was examined using three models The regulation of PRAP1 by differentiation was first studied in relation to the WNT/TCF signaling pathway The WNT/TCF pathway is the main mechanism controlling the cell renewal program of the intestinal epithelium (Pinto and Clevers 2005) Activation of WNT/TCF pathway drives the expression of specific genes that are related to proliferation and found

at the proliferative compartment of crypts On the other hand, genes repressed by WNT/TCF pathway are related to differentiation and found at the top of the crypts and in villi (van de Wetering, Sancho et al 2002) Thus, introduction of N-terminally truncated TCF that inhibits the WNT/TCF pathway can lead to differentiation in an in vitro assay A stable cell line, L8 that expresses a doxycycline-inducible N-terminally truncated TCF-4 protein was used to mimic intestinal differentiation Upon induction by doxycycline, the dnTCF-4 protein effectively blocked the activation of WNT/TCF signaling pathway, leading to cellular differentiation The differentiation status was determined by the expression of a well-characterized intestinal differentiation marker, GALECTIN-

4 Our results from this in vitro differentiation model have shown that inhibition

of WNT/TCF pathway resulted in marked induction of PRAP1 expression, indicating that PRAP1 is regulated by the WNT/TCF pathway

The second model used was the sodium butyrate model Sodium butyrate

is an inhibitor of HDACs Our results showed that PRAP1 expression was highly induced by sodium butyrate The expression of PRAP1 was tightly associated with differentiation with a high positive correlation factor of 0.96 between PRAP1 expression and the differentiation marker alkaline phosphatase Class I histone deacetylases (HDACs) has been previously described to be involved in the regulation of mammalian intestine development and epithelial differentiation (Tou, Liu et al 2004) In their study, PRAP1 was reported to be induced by the inhibition of HDACs This is consistent with our results and our previous data (Zhang, Wong et al 2003), showing regulation of PRAP1 by epigenetic mechanisms including DNA methylation and histone acetylation Since fermentation in the intestinal tract leads to the production of short-chain fatty acids such as sodium butyrate, we hypothesize that PRAP1 may be involved in the homeostasis of intestinal epithelium in response to environmental factors

The third model we used was the glucose deprivation model By culturing cells in glucose free medium, a sub-population of cells (Glu-cells) with the ability

to grow in the absence of glucose was selected These cells were reported to exhibit an enterocytic differentiation (Zweibaum, Pinto et al 1985) Consistent with their report, our results showed that an increase in alkaline phosphatase activity was detected in these Glu-cells as compared to the control cells Our results showed that PRAP1 expression was induced in these better differentiated Glu-cells as compared to the control cells

In conclusion, our results showed that expression of PRAP1 was tightly regulated by differentiation via the intrinsic WNT/TCF signaling pathway, and in response to environmental cues such as fermentation and nutrients

4.1.1.3 Regulation of PRAP1 expression by differentiation

In order to understand the mechanism of PRAP1 regulation by

differentiation, we studied the changes of PRAP1 expression at the mRNA level Our results showed that PRAP1 was induced at the mRNA level by both

WNT/TCF and sodium butyrate differentiation models To explore whether this increase in mRNA was due to an increase in transcription activity, we cloned and

characterized the promoter of PRAP1 We identified a core promoter of PRAP1 located within 200 base pairs upstream of PRAP1 transcription start site The transcriptional regulation of PRAP1 was studied using two constructs, the core

promoter and the longest construct we have cloned that span over 3900 base pairs Interestingly, our results showed induction of differentiation by inhibiting the WNT/TCF pathway did not result in any significant changes in the promoter

activities of PRAP1 However, due to technical limitations in our study, we cannot exclude the possibility that the expression of PRAP1 is regulated at transcriptional

level by regulatory elements that are present further upstream of our longest promoter construct, intronic regions and/or at the 3’UTR It may well be that an interplay of several regulatory elements dispersed over distinct loci contributes to transcriptional regulation of PRAP1, which may prove extremely difficult to

recapitulate in in vitro promoter studies Alternatively, PRAP1 may be regulated

at the post-transcriptional level through stabilization of the PRAP1 transcript

With our available tools, we sought to investigate whether PRAP1 mRNA

transcripts are being stabilized upon treatment with genotoxic agents To test this

hypothesis, we monitored the stability of PRAP1 mRNA over a period of 4 hours

following inhibition of endogenous transcription Our results showed that the

half-life of the PRAP1 transcripts was dramatically enhanced in the WNT/TCF differentiation model The PRAP1 transcripts was only reduced by 10% over a period of 2 hours, whereas in the control cells, PRAP1 transcripts were less stable

and showed 25% degradation and was further reduced by 70% at the 4 hour time point The WNT/TCF signaling pathway has been shown to regulate stability of mRNA in addition to its direct transcriptional control For instance, β-catenin stabilized the cyclooxygenase-2 (COX-2) mRNA by interacting with the AU-richelement in the 3'-untranslated region (3'-UTR, (Lee and Jeong 2006) However,

there is no AU-rich element present in the 3’-UTR of PRAP1 mRNA, suggesting

the involvement of other regulation mechanism(s)

In summary, we have demonstrated that induction of PRAP1 by

differentiation was mediated partly via the stabilization of mRNA Further

investigations are required to examine the regulation of PRAP1 at the

transcriptional level using constructs containing promoter fragments located further upstream, intronic regions and/or 3'UTR, and to elucidate the mechanism

underlying the stabilization of PRAP1 mRNA

4.1.2 Effect of PRAP1 on differentiation

To study the effect of PRAP1 on differentiation, we perturbed the expression of PRAP1 and examined its effect on the differentiation status as measured by established differentiation markers Our results from this study have shown that transient overexpression of PRAP1 did not result in significant changes in the level of alkaline phosphatase activities and GALECTIN-4

PRAP1 alone is not sufficient to induce differentiation In addition, the inhibition

of PRAP1 did not result in a reversal of dnTCF induced differentiation in L8 cells This indicates that PRAP1 gene does not play an essential role in the induction of differentiation of epithelial cells by the WNT/TCF signaling pathway

In conclusion, even though we showed that PRAP1 expression is tightly associated with differentiation, PRAP1 protein by itself is neither necessary nor sufficient in inducing differentiation, at least with the conditions and cell lines used in this study This suggests that PRAP1 is a product of differentiated cells that subserves specific functions such as absorption, digestion and innate immunity

4.1.3 PRAP1 and innate immunity

The detection of PRAP1 in the intestinal epithelial cells and the intermediate-like cells, which secretes human α-defensin 5 (HD-5), suggest that PRAP1 may also be involved in the innate immunity of the intestinal tract Our results showed that PRAP1 protein binds to the surface of bacteria, which was confirmed with two different methods The antibacterial property of PRAP1 was assayed by a commercial company Their initial results showed that the binding of PRAP1 to bacteria was bactericidal with an IC50 (50% of bacterial growth

inhibition) of 5µM (~200µg/ml) against a panel of bacteria, including eight E coli strains, Shigella and S aureus This level of antibacterial activity was comparable

to that of many well-defined antimicrobial peptides For instance, α-defensins in human neutrophils (HNP 1-3) are active at concentrations of higher than 100µg/ml and the bacteriostatic effect of human β-defensin 2 is at more than

100µg/ml for S aureus (Schroder and Harder 1999) The bactericidal activity of

PRAP1 was found to be pH-dependent, consistent with other studies (Pereira,

Erdem et al 1993; Lee, Cho et al 1997) PRAP1 has higher antibacterial activity

at pH 4.5 than at neutral pH The theoretical pI of PRAP1 is 5.2, suggesting that it would acquire a net positive charge at pH 4.5 The increased positive charge may enhance the electrostatic interaction of PRAP1 with the negatively charged phospholipids and lipopolysaccharides on the bacteria outer membrane The increased antibacterial activity at lower pH may suggest a possible role of PRAP1

in the protection of stomach against infection Interestingly, PRAP1 was also found to be expressed more abundantly in the stomach as compared to small intestine and colon (Zhang, Wong et al 2003), further supporting this hypothesis

The PRAP1 protein is neither homologous nor similar to other antibacterial peptides identified In the human gastrointestinal tract, antimicrobial peptides such as α-defensins (HD-5 in small intestine) and β-defensin (hBD-3 in stomach and colon) are the major players These defensins are small (15-20 residues for α-defensin and 38-47 residues for β-defensin), cysteine-rich cationic peptides In contrast, PRAP1 is a relatively larger protein of 131 residues after the cleavage of its signal peptide, and is a cysteine-free anionic protein Moreover, other well-defined anionic antimicrobial peptides are small homopolymeric peptides of 7 aspartic acid residues, and require zinc as a cofactor for bactericidal activity On the other hand, PRAP1 protein only has 9 aspartic acid residues that are distributed randomly throughout the whole protein These suggest that PRAP1 may exert its antimicrobial activity via novel mechanisms

The involvement of PRAP1 in acquired immunity was also examined Our results showed that PRAP1 did not result in any significant changes in the phagocytosis of bacteria by macrophages However, this may due to the high level

of redundancy associated with acquired immunity and the limitation of our assay which showed saturation at the earliest time-point possible

In summary, identification of new antimicrobial proteins is a promising avenue to explore in view of the growing problem of acquired resistance to antibiotics It is believed that the acquired resistance as seen with commonly used antibiotics is unlikely to occur with the use of natural defensins or other antimicrobial peptides This may be due to the different mode of action of antimicrobial peptides Future work is needed to further confirm the antibacterial property of PRAP1 and to elucidate its mechanism of action

Among the possible lines of future work is to demonstrate the bactericidal activity in different preparations of purified recombinant PRAP1 protein and with peptides obtained from more than one commercial source to exclude the possibility of batch specific effect attributable to contaminants introduced during the purification process Moreover, as the majority of known mammalian antimicrobial peptides are produced and stored as precursors in granules of Paneth cells, and processed to their mature form by trypsin digestion, the antimicrobial activity of PRAP1 may be regulated in a similar manner A logical next step would be to study the antimicrobial activity of various PRAP1 fragments following proteolytic cleavage with trypsin Identification of PRAP1 peptides in the lumen of mouse intestines would provide in vivo evidence for this hypothesis

It would also be interesting to determine if PRAP1 acts through the same mechanism as defensins in permeabilizing the membrane of bacteria, or through

other novel mechanisms