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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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Effects of p38b MAPK knockdown in ISCs and EBs on PVR-induced phenotypes.                                            (A) Effect of D-p38b knockdown in ISCs/EBs on ectopic PVR-induced                                            ISC proliferation. The PH3-positive cells in the midgut of the 3-day-old                                            flies were counted. White bar, esg>+; black bar, esg>UAS-PVR;                                            dark gray bar, esg>UAS-D-p38bas;UAS-PVR; gray                                            bar, esg>UAS-D-p38bas. The number of PH3-positive                                            cells detected per midgut of esg>+ flies was set as 1. P-values                                            were calculated using Student's t-test. (B) Effect of D-p38b                                            knockdown in ISCs and EBs on PVR-induced accumulation of large esg- and                                            Delta-positive cells. The guts of 3-day-old flies were labeled with                                            anti-Delta and anti-GFP. (a-a''), esg>+; (b-b''), esg>UAS-PVR;                                            (c-c''), esg>UAS-D-p38bas;UAS-PVR; (d-d''), esg>UAS-D-p38bas.                                            Overlay (DAPI, blue; anti-Delta, red; anti-GFP, green). Asterisk indicates                                            EC-like large esg-positive cell. Arrow indicates Delta-positive cells.                                            Scale bar, 5 μM. C. Effect of                                            D-p38b knockdown in ISCs and EBs on PVR-induced Delta mRNA expression.                                            Expression of Delta was measured by quantitative RT-PCR of dissected gut                                            from 3-day-old flies. White bar, esg>+; black bar, esg>UAS-PVR;                                            dark gray bar, esg>UAS-D-p38bas;UAS-PVR; gray                                            bar, esg>UAS-D-p38bas. Expression was normalized to                                            the expression of rp49. The level of Delta mRNA in the midgut of esg>+                                            flies was set as 1. P-values were determined using Student's t-test.
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Figure 6: Effects of p38b MAPK knockdown in ISCs and EBs on PVR-induced phenotypes. (A) Effect of D-p38b knockdown in ISCs/EBs on ectopic PVR-induced ISC proliferation. The PH3-positive cells in the midgut of the 3-day-old flies were counted. White bar, esg>+; black bar, esg>UAS-PVR; dark gray bar, esg>UAS-D-p38bas;UAS-PVR; gray bar, esg>UAS-D-p38bas. The number of PH3-positive cells detected per midgut of esg>+ flies was set as 1. P-values were calculated using Student's t-test. (B) Effect of D-p38b knockdown in ISCs and EBs on PVR-induced accumulation of large esg- and Delta-positive cells. The guts of 3-day-old flies were labeled with anti-Delta and anti-GFP. (a-a''), esg>+; (b-b''), esg>UAS-PVR; (c-c''), esg>UAS-D-p38bas;UAS-PVR; (d-d''), esg>UAS-D-p38bas. Overlay (DAPI, blue; anti-Delta, red; anti-GFP, green). Asterisk indicates EC-like large esg-positive cell. Arrow indicates Delta-positive cells. Scale bar, 5 μM. C. Effect of D-p38b knockdown in ISCs and EBs on PVR-induced Delta mRNA expression. Expression of Delta was measured by quantitative RT-PCR of dissected gut from 3-day-old flies. White bar, esg>+; black bar, esg>UAS-PVR; dark gray bar, esg>UAS-D-p38bas;UAS-PVR; gray bar, esg>UAS-D-p38bas. Expression was normalized to the expression of rp49. The level of Delta mRNA in the midgut of esg>+ flies was set as 1. P-values were determined using Student's t-test.

Mentions: 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 Delta-positive 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.


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)

Effects of p38b MAPK knockdown in ISCs and EBs on PVR-induced phenotypes.                                            (A) Effect of D-p38b knockdown in ISCs/EBs on ectopic PVR-induced                                            ISC proliferation. The PH3-positive cells in the midgut of the 3-day-old                                            flies were counted. White bar, esg>+; black bar, esg>UAS-PVR;                                            dark gray bar, esg>UAS-D-p38bas;UAS-PVR; gray                                            bar, esg>UAS-D-p38bas. The number of PH3-positive                                            cells detected per midgut of esg>+ flies was set as 1. P-values                                            were calculated using Student's t-test. (B) Effect of D-p38b                                            knockdown in ISCs and EBs on PVR-induced accumulation of large esg- and                                            Delta-positive cells. The guts of 3-day-old flies were labeled with                                            anti-Delta and anti-GFP. (a-a''), esg>+; (b-b''), esg>UAS-PVR;                                            (c-c''), esg>UAS-D-p38bas;UAS-PVR; (d-d''), esg>UAS-D-p38bas.                                            Overlay (DAPI, blue; anti-Delta, red; anti-GFP, green). Asterisk indicates                                            EC-like large esg-positive cell. Arrow indicates Delta-positive cells.                                            Scale bar, 5 μM. C. Effect of                                            D-p38b knockdown in ISCs and EBs on PVR-induced Delta mRNA expression.                                            Expression of Delta was measured by quantitative RT-PCR of dissected gut                                            from 3-day-old flies. White bar, esg>+; black bar, esg>UAS-PVR;                                            dark gray bar, esg>UAS-D-p38bas;UAS-PVR; gray                                            bar, esg>UAS-D-p38bas. Expression was normalized to                                            the expression of rp49. The level of Delta mRNA in the midgut of esg>+                                            flies was set as 1. P-values were determined using Student's t-test.
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Figure 6: Effects of p38b MAPK knockdown in ISCs and EBs on PVR-induced phenotypes. (A) Effect of D-p38b knockdown in ISCs/EBs on ectopic PVR-induced ISC proliferation. The PH3-positive cells in the midgut of the 3-day-old flies were counted. White bar, esg>+; black bar, esg>UAS-PVR; dark gray bar, esg>UAS-D-p38bas;UAS-PVR; gray bar, esg>UAS-D-p38bas. The number of PH3-positive cells detected per midgut of esg>+ flies was set as 1. P-values were calculated using Student's t-test. (B) Effect of D-p38b knockdown in ISCs and EBs on PVR-induced accumulation of large esg- and Delta-positive cells. The guts of 3-day-old flies were labeled with anti-Delta and anti-GFP. (a-a''), esg>+; (b-b''), esg>UAS-PVR; (c-c''), esg>UAS-D-p38bas;UAS-PVR; (d-d''), esg>UAS-D-p38bas. Overlay (DAPI, blue; anti-Delta, red; anti-GFP, green). Asterisk indicates EC-like large esg-positive cell. Arrow indicates Delta-positive cells. Scale bar, 5 μM. C. Effect of D-p38b knockdown in ISCs and EBs on PVR-induced Delta mRNA expression. Expression of Delta was measured by quantitative RT-PCR of dissected gut from 3-day-old flies. White bar, esg>+; black bar, esg>UAS-PVR; dark gray bar, esg>UAS-D-p38bas;UAS-PVR; gray bar, esg>UAS-D-p38bas. Expression was normalized to the expression of rp49. The level of Delta mRNA in the midgut of esg>+ flies was set as 1. P-values were determined using Student's t-test.
Mentions: 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 Delta-positive 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.

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