The role of p38b MAPK in age-related modulation of intestinal stem cell proliferation and differentiation in Drosophila.

It is important to understand how age-related changes in intestinal stem cells (ISCs) may contribute to age-associated intestinal diseases, including cancer. Drosophila midgut is an excellent model system for the study of ISC proliferation and differentiation. Recently, age-related changes in the Drosophila midgut have been shown to include an increase in ISC proliferation and accumulation of mis-differentiated ISC daughter cells. Here, we show that the p38b MAPK pathway contributes to the age-related changes in ISC and progenitor cells in Drosophila. D-p38b MAPK is required for an age-related increase of ISC proliferation. In addition, this pathway is involved in age and oxidative stress-associated mis-differentiation of enterocytes and upregulation of Delta, a Notch receptor ligand. Furthermore, we also show that D-p38b acts downstream of PVF2/PVR signaling in these age-related changes. Taken together, our findings suggest that p38 MAPK plays a crucial role in the balance between ISC proliferation and proper differentiation in the adult Drosophila midgut.


INTRODUCTION
In mammals, intestinal homeostasis is maintained by the balance between intestinal stem cell (ISC) proliferation, directed differentiation, and removal of dead cells in adults [1]. The precise mechanism by which proliferation and differentiation of stem cells is lost with age and/or oxidative stress is unknown. These effects on stem cells result in age-related diseases, such as cancer [2,3]. Therefore, it is important to understand how agerelated changes in ISC proliferation and differentiation contribute to age-associated intestinal diseases, including cancer. Recently, Drosophila melanogaster was shown to be an excellent model system for the study of ISC biology and aging. It was demonstrated that proliferating progenitor cells reside within the intestinal epithelium of adult Drosophila, similar to vertebrate intestine [4,5]. Adult Drosophila midgut cells can be identified as ISCs, enteroblasts (EBs), enteroendocrine large esg-positive cells. In addition they showed that the age-related changes are associated with aberrant Delta/Notch and JNK signaling [8]. More recently, involvement of the insulin receptor and the JAK-STAT signaling pathways was shown in ISC proliferation in response to tissue damage and during immune response, respectively [9,10].
In mammals, it has been well demonstrated that p38 MAPK is activated in response to various physical and chemical stresses, such as oxidative stress, UV irradiation, hypoxia and ischemia [11]. Fu et al. reported increased p38 MAPK activation in ISCs and their daughter cells in the small intestine after ischemia [12]. In Drosophila, two p38 MAPK isoforms have been identified, D-p38a and D-p38b. D-p38s are activated by various stresses, including UV, lipopolysaccharide (LPS), and osmotic stress [13,14]. It was reported that D-p38b mRNA levels were detected in the developing posterior midgut [14]. We previously observed an increase in expression of a D-p38b-lacZ reporter construct in the posterior midgut by septic injury [15]. However, the role of p38 MAPK in ISC proliferation and differentiation remains unknown.
In the current study, we tested whether D-p38b MAPK is involved in age and oxidative stress-associated modulation of ISC proliferation and differentiation in the adult Drosophila midgut. We further examined the serial relationship between D-p38b MAPK and PVR signaling in age-associated intestinal changes.

Increased expression of a D-p38b-lacZ reporter construct in ISCs and progenitors of aged midgut
To determine the role of D-p38b MAPK in age-related changes in the adult Drosophila midgut, we first investigated D-p38b expression in the adult midgut using transgenic flies carrying a D-p38b-lacZ reporter construct [15]. Increased expression of the D-p38b-lacZ reporter construct in aged midgut was detected by X-gal staining ( Figure 1A). In Figure 1B, we analyzed the expression pattern of D-p38b-lacZ in the aged guts using anti-β-gal antibody and anti-Delta antibody, a marker of ISC [4,5]. Interestingly, the expression pattern of D-p38b-lacZ in young and aged posterior midguts indicates that the expression of D-p38b-lacZ increases in ISCs and neighboring cells in the adult Drosophila midgut.  www.impactaging.com

D-p38b MAPK is required for the age-related increase in ISC proliferation
We previously demonstrated an age-related increase of ISC proliferation in adult Drosophila midgut via BrdU incorporation and PH3 cell staining [7]. Therefore, we investigated whether D-p38b MAPK is involved in the age-related increase in proliferation of ISCs. To determine activation of D-p38b in ISCs, we used UAS-D-p38b + or UAS-D-p38b as [16] and esg-GAL4 flies, which express GAL4 and UAS-GFP in midgut ISCs and EBs [4]. Adachi-Yamada et al. reported that overexpression of UAS-D-p38b + , under control of hs-GAL4, increased phosphorylation and enzymatic activity of D-p38b. In addition they demonstrated that overexpression of the UAS-D-p38b as construct, which contains the inverted cDNA, of full-length D-p38b, suppressed the ectopic tkv-induced wing phenotype [16]. Guts from 30-day-old wild-type flies, esg>+, showed a significant increase in the number of BrdUlabeled large and small cells compared to those from 5day-old flies ( Figure 2A, panels a and b). This is consistent with our previous study [7]. However, guts expressing D-p38b as in ISCs and EBs by esg-GAL4 showed no age-related increase in DNA synthesis in the posterior midgut (Figure 2A, panels c and d). We also observed no age-related increase in DNA synthesis in the posterior midgut from two D-p38b mutants ( Figure  2A, panels e-h). Flies overexpressing D-p38b + under the control of esg-GAL4 had increased numbers of BrdUlabeled midgut cells compared to wild-type flies at 5days-old (Supplementary Figure S1). We next analyzed the role of D-p38b MAPK in ISC division with anti-PH3 antibody, which detects only proliferating cells [4,5]. In consistent with our previous study [7], we observed an age-related increase in the number of PH3positive cells in 5-, 30-, and 60-day-old guts in flies carrying one copy of esg-GAL4 ( Figure 2B). However, expression of D-p38b as in ISCs and EBs by esg-GAL4 suppressed the age-related increase in cellular division of ISCs ( Figure 2B). We also analyzed the number of PH3-positive cells in the aged guts of two D-p38b mutants. The number of PH3-positive cells in both mutants decreased with age ( Figure 2B). As expected, the guts from 5-day-old flies overexpressing D-p38b + under the control of esg-GAL4 showed a 2.6-fold increase in the number of PH3-positive cells compared to wild-type ( Figure 2C). To confirm the role of D-p38b MAPK in proliferation of ISCs, we generated green fluorescent protein (GFP)-marked clones overexpressing D-p38b + or D-p38b as using Flp-out cassette [17] and counted the cell number per one GFP-positive cluster in the posterior midgut. The size of the colony indicates the rate of cell division of the ISCs [4]. While most colonies in the control guts contained 4-7 cells ( Figure 3A and B, black circle), clones in the guts of D-p38b as were composed of 1-4 cells ( Figure 3A and B, red triangle). Most clones expressing ectopic D-p38b contained 9-13 cells ( Figure 3A and B, blue circle). Collectively, these data indicate that D-p38b is involved in ISC division and is required for the age-related increase in ISC proliferation.

D-p38b MAPK is involved in age-related changes of ISC and progenitor cell differentiation
To assess whether D-p38b MAPK is required for agerelated changes in ISC and progenitor cell differentiation, we analyzed the ratio of Su(H)GBEpositive to total cells to determine the frequency of EBs differentiation to ECs. It was reported that high Su(H)GBE-positive EBs become ECs [6]. Consistent with our previous study, the number of Su(H)GBEpositive cells in the guts of control esg>Su(H)GBE-lacZ flies increased with age ( Figure 4A) [7]. In contrast, the number of Su(H)GBE-positive cells in the guts of 30day-old esg>UAS-D-p38b as ;Su(H)GBE-lacZ flies was 0.5-fold less than that of 5-day-old flies ( Figure 4A). Figure S3A). We also examined the ratio of Prosperopositive cells, to determine the frequency of EBs differentiation to EEs. It was reported that the ratio of EEs to total cells does not change [8]. Interestingly, in guts expressing D-p38b as in ISCs and EBs, the ratio of EE to total cells increased with age. A 1.48-fold increase was observed in 30-day-old compared to 5day-old esg>UAS-D-p38b as ;Su(H)GBE-lacZ flies, while of the ratio of EE to total cells did not change in control flies ( Figure 4B). Esg-positive cells had a spherical cell shape in the guts of esg>UAS-D-p38b as , distinguishing them morphologically from their angularly shaped counterparts in the guts of esg>+ ( Figure 4C). We also detected that the ratio of EE to total cells in the 30-dayold posterior midgut of two D-p38b mutant flies was 2.2-fold and 2.1-fold higher than that in the 30-day-old gut of control flies (Supplementary Figure S2A and B).

Overexpression of D-p38b + in ISCs and EBs resulted in an increased number of Su(H)GBE-positive cells compared to control flies at 5-days-old (Supplementary
These results indicate that D-p38b MAPK is required for the age-related increase in ISC differentiation to ECs in the adult posterior midgut. Next, to determine whether D-p38b MAPK is involved in the terminal differentiation defect of ECs in aged guts, we analyzed the ratio of ECs to Su(H)GBEpositive cells. In the guts of control flies, the ratio of ECs to Su(H)GBE-positive cells at 30-days-old decreased by 0.5-fold compared to that at 5-days-old. In contrast, the ratio in the guts of flies expressing D-p38b www.impactaging.com anti-sense in ISCs and EBs at 30-days-old was 1.45-fold higher than at 5-days-old ( Figure 4C). The ratio of ECs to Su(H)GBE-positive cells in 5-day-old guts of esg>UAS-D-p38b + ;Su(H)GBE-lacZ was slightly lower than those of control flies (Supplementary Figure S3B).
These results indicate that D-p38b MAPK is involved in the defect of EC differentiation in aged guts.
We also analyzed the effects of D-p38b MAPK activation on the morphology of ISCs and EBs in the gut. We observed age-related accumulation of EC-like large esg-positive cells in the guts of control flies, esg>Su(H)GBE-lacZ ( Figure 4D, panels a and d).
Interestingly, age-related accumulation of EC-like large esg-positive cells was not detected in the guts of esg>UAS-D-p38b as ;Su(H)GBE-lacZ flies ( Figure 4E, panels g and j). D-p38b + overexpression in ISCs and EBs induced EC-like large esg-positive cells at 5-days-old (Supplementary Figure S3C). These results indicate that D-p38b MAPK is involved in age-related accumulation of EC-like large esg-positive cells in the posterior midgut.
We observed a 4-fold and 3.4-fold increase of Delta mRNA in 30-day-old guts compared to 5-day-old guts of control flies, Oregon-R and esg>+, respectively ( Figure  4F). We examined whether D-p38b MAPK is involved in age-related modulation of Delta expression within the gut. Expectantly, Delta mRNA levels in the gut of esg>UAS-D-p38b as flies did not change with age ( Figure  4F). Two D-p38b mutants also showed no age-related change in Delta expression ( Figure 4F). Overexpression of D-p38b + in ISCs and EBs by esg-GAL4 increased Delta expression up to 1.5-fold compared to control flies at 5-days-old ( Figure 4G). This indicates that D-p38b is involved in the regulation of Delta expression.

D-p38b MAPK is involved in oxidative stressinduced differentiation defects
To examine whether D-p38b MAPK is involved in oxidative stress-induced mis-differentiation of ISCs and progenitors, we analyzed the morphology of esg-or Su(H)GBE-positive cells in guts expressing D-p38b as in ISCs and EBs after paraquet (PQ) exposure. EC-like large esg-and Su(H)GBE-positive cells accumulated in the guts of 5-day-old esg>Su(H)GBE-lacZ flies after 10 mM PQ treatment ( Figure 5A, panels a-f). In contrast, the guts of esg>UAS-D-p38b as ;Su(H)GBE-lacZ flies showed no oxidative stress-induced accumulation of large esgand Su(H)GBE-positive cells ( Figure 5A, panels g-l). We also observed that expression of D-p38b as in ISCs and EBs suppressed the oxidative stress-induced increase of Delta mRNA level in guts via quantitative real-time PCR ( Figure 5B). These results indicate that D-p38b MAPK is involved in oxidative stress-induced mis-differentiation of ISCs and progenitors.  www.impactaging.com

D-p38b is downstream of PVF2/PVR signaling in intestinal epithelium
We previously reported that PVF2/PVR signaling contributes to an age and oxidative stress-related increase in stem cell proliferation, and a defect in differentiation of ECs [7]. To investigate whether D-p38b MAPK can act downstream of PVF2/PVR signaling in the age-related changes of intestinal epithelium, we first analyzed the effect of D-p38b knockdown on the ISC division. PVR overexpression in ISCs and EBs using esg-GAL4 driver increases ISC division [7]. However, D-p38b knockdown in ISCs and EBs partially suppressed the PVR-induced increase of PH3-positive cells ( Figure 6A). These results indicate that D-p38b MAPK acts downstream of PVR signaling in midgut ISC proliferation. We next investigated whether D-p38b MAPK is related to PVR signaling in the mis-differentiation of ISCs and progenitors. We observed accumulation of EC-like large esg-positive cells in 5-day-old guts of esg>UAS-PVR flies ( Figure  6B, panels b-b''). Interestingly, PVR-induced increase in the number of EC-like esg-positive cells was partially suppressed by D-p38b knockdown in ISCs and EBs ( Figure 6B, panel c and c''). Overexpression of PVR in ISCs and EBs results in accumulation of aberrant Deltapositive cells ( Figure 6B. panel b'). These cells have high Delta levels and the phenotype was partially suppressed by D-p38b knockdown in ISCs and EBs ( Figure 6B, panel c'). In addition, expression of PVR in ISCs and EBs induced a 4-fold increase in Delta expression compared to control flies, which is partially suppressed by D-p38b knockdown ( Figure 6C). These results indicate that D-p38b MAPK acts downstream of PVR signaling in the mis-differentiation of ISCs and progenitors.

DISCUSSION
In a previous study, we reported age-related changes including an increase in stem cell proliferation and a defect in stem and progenitor cell differentiation to EC. In addition we demonstrated that a PDGF/VEGF-like growth factor, PVF2/PVR, is involved in the age and oxidative stress-associated modulation in the Drosophila midgut [7]. Recently, Biteau et al. also reported the phenomenon of aged intestinal epithelium due to aberrant JNK activity [8]. Here, we investigated whether D-p38b MAPK activity is required for age and oxidative stress-associated changes in intestinal epithelium.
We found an age-related increase of a D-p38b reporter construct in ISCs and EBs in the adult Drosophila midgut. In mammals, it was reported that activated p38 MAPK was detected in the ISCs and their daughter cells after ischemia [12]. We analyzed whether D-p38b MAPK plays a role in the age-related increase of ISC proliferation in Drosophila midgut. Flies expressing D-p38b as in ISCs and EBs and two D-p38b mutants did not show an age-related increase of ISC proliferation. Previously, we reported that PVF2/PVR signaling is involved in an age and oxidative stress-related increase in ISC proliferation [7]. Here, we showed that PVRinduced increase in ISC proliferation was suppressed by D-p38b MAPK knockdown in ISCs and EBs. These data suggest that D-p38b MAPK acts downstream of PVR signaling in ISC proliferation. However, it should be noted that D-p38b expression in ISCs and EBs partially suppressed the PVR-induced ISC proliferation.
Recently, Biteau et al. reported that JNK activity contributes to the age-associated increase in stem cell proliferation [8]. It was reported that Wg signaling, insulin receptor signaling pathway, and the JAK-STAT pathway are involved in ISC proliferation in the Drosophila adult midgut [9,10,17]. Jiang and Edgar reported that EGFR/RAS/ERK pathway is required for proliferation of adult midgut progenitors [18]. Therefore, cross-talk between these signaling pathways and PVR-p38 MAPK signaling in the regulation of ISC proliferation would be an interesting subject to investigate.
In a previous study, we also found that the number of EBs differentiating to ECs increased with age [7]. Ohlstein and Spradling reported that ISC within the adult midgut normally produce EB via asymmetric division. Most EBs differentiate to ECs, while a few differentiate to EEs [6]. It was reported that the ratio of EE to total cells did not change in the midgut with age [8]. These data suggest that there is an age-related increase in the number of EBs giving rise to ECs. Here, we showed that loss of D-p38 signaling suppressed the age-related increase of Su(H)GBE-positive cells and increased the ratio of EE to total cells, especially in old flies. In addition, the esg-positive cells with aberrant D-p38 signaling were spherical instead of angular shaped. Recently, Maeda et al. reported that knockdown of Ecad in the gut resulted in esg-positive cells with a spherical cell shape and an increase of the number of EEs [19]. Our data suggest that D-p38b MAPK is required for ISC commitment to EB differentiation to EC.
Recently, it was reported accumulation of aberrant EClike esg-positive cells is an indicator for misdifferentiation of ECs. It was also proposed that aberrant expression of Delta in ISCs and progenitors is associated with the accumulation of large esg-positive cells [8]. In this study, expression of D-p38b as in ISCs www.impactaging.com and EBs suppressed the age-related and oxidative stress-induced accumulation of EC-like esg-positive cells, and partially suppressed the mis-differentiation induced by ectopic PVR. Furthermore, loss of D-p38b MAPK signaling in ISCs and EBs suppressed age and stress-induced Delta expression, and partially suppressed PVR-induced Delta expression. These data suggest a cross-talk between PVR and D-p38b MAPK signaling in the regulation of Delta expression. Furthermore, our data suggest that D-p38b MAPK acts downstream of PVR signaling, and aberrant D-p38b MAPK signaling results in mis-differentiation in the midgut.
It was reported that in wild-type gut, ECs turnover approximately within one week [5]. Interestingly, we observed that D-p38b knockdown in ECs likely results in cell turnover longer than one week. We also detected that EC size in the guts of flies expressing D-p38b as in ISCs and EBs was larger than those of controls with age (data not shown), suggesting that D-p38b MAPK may be involved in EC death. Recent studies in mammals demonstrated that p38 MAPK is involved in intestinal epithelial cell apoptosis through activation of Bax in stress conditions [20,21].
In light of these findings, we propose that D-p38b MAPK plays a crucial role in the balance between regeneration of intestinal epithelium and proper differentiation.
The All experiments were conducted and evidenced similar results in both females and males. The results described in this present study were obtained from the females.
Oligonucleotides. Oligonucleotide primers of rp49 and Delta were previously described [8]. All oligonucleotides were chemically synthesized.