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

Park JS, Kim YS, Yoo MA - Aging (Albany NY) (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Pusan National University, Busan 609-735, Korea.

ABSTRACT
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.

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Effect of D-p38b MAPK signaling on DNA synthesis of intestinal cells and ISC division.                                                (A) Effects of D-p38b MAPK modulation on BrdU incorporation levels                                                in the adult midgut. Twenty-five day-old flies expressing esg>+ (a                                                and b), esg>UAS-D-p38bas(c and b), p38bEY11174                                                                (e and f) or p38bKG01337 (g and h) were fed on                                                0.2 mg/ml BrdU media for 4 days, and stained with anti-BrdU. Overlay (DAPI,                                                blue; anti-BrdU, red). Asterisk indicates enlarged EC nuclei. Arrow                                                indicates small ISC, EB or EE cell nuclei. Scale bar, 5 μM. Original                                                magnification is 400x. (B) Effect of D-p38b MAPK activity on the                                                number of PH3-positive cells within the adult gut. Number of PH3-positive                                                cells detected per midgut of 5-, 30- and 60-day-old esg>+ or                                                        esg>UAS-D-p38bas flies and 3- and 30-day-old control                                                flies, p38bEY11174 or p38bKG01337. The                                                number of PH3-positive cells detected per midgut of 5-day-old flies was set                                                as 1. White bar, 5-day-old flies; gray bar, 30-day-old flies; black bar,                                                60-day-old flies. P-values were calculated using Student's t-test. (C)                                                Effect of D-p38b MAPK activation on the number of PH3-positive cells.                                                Number of PH3-positive cells in the midguts of 5-day-old flies carrying esg>+                                                or esg>UAS-D-p38b+ were analyzed. White bar, esg>+;                                                gray bar, esg>UAS-D-p38b+. The number of PH3-positive                                                cells detected per midgut of 5-day-old flies was set as 1. P-values were                                                calculated using Student's t-test.
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Figure 2: Effect of D-p38b MAPK signaling on DNA synthesis of intestinal cells and ISC division. (A) Effects of D-p38b MAPK modulation on BrdU incorporation levels in the adult midgut. Twenty-five day-old flies expressing esg>+ (a and b), esg>UAS-D-p38bas(c and b), p38bEY11174 (e and f) or p38bKG01337 (g and h) were fed on 0.2 mg/ml BrdU media for 4 days, and stained with anti-BrdU. Overlay (DAPI, blue; anti-BrdU, red). Asterisk indicates enlarged EC nuclei. Arrow indicates small ISC, EB or EE cell nuclei. Scale bar, 5 μM. Original magnification is 400x. (B) Effect of D-p38b MAPK activity on the number of PH3-positive cells within the adult gut. Number of PH3-positive cells detected per midgut of 5-, 30- and 60-day-old esg>+ or esg>UAS-D-p38bas flies and 3- and 30-day-old control flies, p38bEY11174 or p38bKG01337. The number of PH3-positive cells detected per midgut of 5-day-old flies was set as 1. White bar, 5-day-old flies; gray bar, 30-day-old flies; black bar, 60-day-old flies. P-values were calculated using Student's t-test. (C) Effect of D-p38b MAPK activation on the number of PH3-positive cells. Number of PH3-positive cells in the midguts of 5-day-old flies carrying esg>+ or esg>UAS-D-p38b+ were analyzed. White bar, esg>+; gray bar, esg>UAS-D-p38b+. The number of PH3-positive cells detected per midgut of 5-day-old flies was set as 1. P-values were calculated using Student's t-test.

Mentions: 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-p38bas [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-p38bas 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 BrdU-labeled large and small cells compared to those from 5-day-old flies (Figure 2A, panels a and b). This is consistent with our previous study [7]. However, guts expressing D-p38bas 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 BrdU-labeled midgut cells compared to wild-type flies at 5-days-old (Supplementary Figure 1). 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 PH3-positive cells in 5-, 30-, and 60-day-old guts in flies carrying one copy of esg-GAL4 (Figure 2B). However, expression of D-p38bas in ISCs and EBs by esg-GAL4 suppressed the age-related increase in cellular division of ISCs (Figure2B). 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 over-expressing D-p38b+ or D-p38bas 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-p38bas 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.


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

Park JS, Kim YS, Yoo MA - Aging (Albany NY) (2009)

Effect of D-p38b MAPK signaling on DNA synthesis of intestinal cells and ISC division.                                                (A) Effects of D-p38b MAPK modulation on BrdU incorporation levels                                                in the adult midgut. Twenty-five day-old flies expressing esg>+ (a                                                and b), esg>UAS-D-p38bas(c and b), p38bEY11174                                                                (e and f) or p38bKG01337 (g and h) were fed on                                                0.2 mg/ml BrdU media for 4 days, and stained with anti-BrdU. Overlay (DAPI,                                                blue; anti-BrdU, red). Asterisk indicates enlarged EC nuclei. Arrow                                                indicates small ISC, EB or EE cell nuclei. Scale bar, 5 μM. Original                                                magnification is 400x. (B) Effect of D-p38b MAPK activity on the                                                number of PH3-positive cells within the adult gut. Number of PH3-positive                                                cells detected per midgut of 5-, 30- and 60-day-old esg>+ or                                                        esg>UAS-D-p38bas flies and 3- and 30-day-old control                                                flies, p38bEY11174 or p38bKG01337. The                                                number of PH3-positive cells detected per midgut of 5-day-old flies was set                                                as 1. White bar, 5-day-old flies; gray bar, 30-day-old flies; black bar,                                                60-day-old flies. P-values were calculated using Student's t-test. (C)                                                Effect of D-p38b MAPK activation on the number of PH3-positive cells.                                                Number of PH3-positive cells in the midguts of 5-day-old flies carrying esg>+                                                or esg>UAS-D-p38b+ were analyzed. White bar, esg>+;                                                gray bar, esg>UAS-D-p38b+. The number of PH3-positive                                                cells detected per midgut of 5-day-old flies was set as 1. P-values were                                                calculated using Student's t-test.
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Figure 2: Effect of D-p38b MAPK signaling on DNA synthesis of intestinal cells and ISC division. (A) Effects of D-p38b MAPK modulation on BrdU incorporation levels in the adult midgut. Twenty-five day-old flies expressing esg>+ (a and b), esg>UAS-D-p38bas(c and b), p38bEY11174 (e and f) or p38bKG01337 (g and h) were fed on 0.2 mg/ml BrdU media for 4 days, and stained with anti-BrdU. Overlay (DAPI, blue; anti-BrdU, red). Asterisk indicates enlarged EC nuclei. Arrow indicates small ISC, EB or EE cell nuclei. Scale bar, 5 μM. Original magnification is 400x. (B) Effect of D-p38b MAPK activity on the number of PH3-positive cells within the adult gut. Number of PH3-positive cells detected per midgut of 5-, 30- and 60-day-old esg>+ or esg>UAS-D-p38bas flies and 3- and 30-day-old control flies, p38bEY11174 or p38bKG01337. The number of PH3-positive cells detected per midgut of 5-day-old flies was set as 1. White bar, 5-day-old flies; gray bar, 30-day-old flies; black bar, 60-day-old flies. P-values were calculated using Student's t-test. (C) Effect of D-p38b MAPK activation on the number of PH3-positive cells. Number of PH3-positive cells in the midguts of 5-day-old flies carrying esg>+ or esg>UAS-D-p38b+ were analyzed. White bar, esg>+; gray bar, esg>UAS-D-p38b+. The number of PH3-positive cells detected per midgut of 5-day-old flies was set as 1. P-values were calculated using Student's t-test.
Mentions: 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-p38bas [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-p38bas 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 BrdU-labeled large and small cells compared to those from 5-day-old flies (Figure 2A, panels a and b). This is consistent with our previous study [7]. However, guts expressing D-p38bas 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 BrdU-labeled midgut cells compared to wild-type flies at 5-days-old (Supplementary Figure 1). 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 PH3-positive cells in 5-, 30-, and 60-day-old guts in flies carrying one copy of esg-GAL4 (Figure 2B). However, expression of D-p38bas in ISCs and EBs by esg-GAL4 suppressed the age-related increase in cellular division of ISCs (Figure2B). 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 over-expressing D-p38b+ or D-p38bas 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-p38bas 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.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Pusan National University, Busan 609-735, Korea.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus