The anti-aging effect of vitamin D and vitamin D receptor in Drosophila midgut

Results

VDR knockdown in ECs induces ISC proliferation

To investigate the role of VDR in the adult Drosophila intestine, flies with ISC/EB-, EB-, EC-, or EE-specific VDR knockdown were generated using flies with the esg^ts>GFP, Su^ts>GFP, Myo^ts>GFP, or pros^ts>GFP genotypes. esg-GFP and PH3 signals were examined, and the number of esg-GFP⁺ and PH3⁺ cells in the guts of esg^ts>GFP and esg^ts>GFP+VDRRi flies kept at 29° C for 7 days did not significantly differ (Figure 1A, 1^st panel and 1B a). In addition, Su-GFP and PH3 signals were examined, and the number of PH3⁺ cells in the guts of Su^ts>GFP and Su^ts>GFP+VDRRi flies kept at 29° C for 7 days did not significantly differ (Figure 1B b); however, the number of Su-GFP⁺ cells significantly decreased (Figure 1A, 2^nd panel). In flies with the Myo^ts>GFP or pros^ts>GFP genotypes, a dramatic increase in the number of PH3+ cells was detected (Figure 1A 3^rd and 4^th panel, and 1B c and d). Quantification of the number of PH3⁺ cells per gut revealed values of 6.58 in the esg^ts>GFP+VDRRi flies (N =14, n = 92; N indicates the number of guts, and n indicates the number of PH3⁺ cells), 4.85 in the esg^ts>GFP flies (N = 13, n = 36) (Figure 1B a), 5.7 in the Su^ts>GFP+VDRRi flies (N =14, n = 80), 6.36 in the Su^ts>GFP flies (N = 14, n = 89) (Figure 1B b), 108.57 in the Myo^ts>GFP+VDRRi flies (N =14, n = 1520), 7.43 in the Myo^ts>GFP flies (N = 14, n = 104) (Figure 1B c), 49.86 in the pros^ts>GFP+VDRRi flies (N =14, n = 698), and 12 in the pros^ts>GFP flies (N = 14, n = 168) (Figure 1B d). These results indicate that the EC-specific knockdown of VDR markedly increases ISC proliferation.

Figure 1. Effects of intestinal cell type-specific VDR knockdown on ISC proliferation. (A, B) ISC and EB (esg^ts), EB (Su^ts), EC (Myo^ts), and EE (pros^ts)-specific knockdown of VDR induces ISC division. Flies carrying esg^ts>GFP and esg^ts>GFP+VDRRi, Su^ts>GFP and Su^ts>GFP+VDRRi, Myo^ts>GFP and Myo^ts>GFP+VDRRi, or pros^ts>GFP and pros^ts>GFP+VDRRi genotypes were cultured for 4 days at 29° C. The midguts of the flies were dissected, fixed, and labeled with anti-PH3 (red) and anti-GFP (green) antibodies and DAPI (blue). The original magnification is 200×. (B) The number of PH3⁺ cells in the midgut with ISC and EB- (esg^ts), EB- (Su^ts), EC- (Myo^ts), and EE (pros^ts)-specific VDR knockdown. Gut samples of esg^ts>GFP and esg^ts>GFP+VDRRi (a), Su^ts>GFP and Su^ts>GFP+VDRRi (b), Myo^ts>GFP and Myo^ts>GFP+VDRRi (c), or pros^ts>GFP and pros^ts>GFP+VDRRi (d) flies maintained at 29° C for a week were labeled with anti-PH3 (red) and anti-GFP (green) antibodies and DAPI (blue). PH3⁺ cell numbers were determined in the entire gut under a microscope. P-values were determined using Student’s t-test. p < 0.001, p < 0.0001. n.s., no significant differences.

VDR knockdown in ECs induces EC death

To assess the role of VDR in EC death, cleaved caspase-3 signals were examined; they were increased in the Myo-GFP⁺ cells of the guts of Myo^ts>GFP and Myo^ts>GFP+VDRRi flies kept at 29° C for 4 days. Very weak cleaved caspase-3 signals were detected in the ECs of Myo^ts>GFP flies (Figure 2A). In contrast to the signal in wild-type Myo^ts>GFP flies, the cleaved caspase-3 signal was markedly increased in the Myo⁺ cells (ECs) of Myo^ts>GFP+VDRRi flies (Figure 2A). Subsequently, the ratio of cleaved caspase-3⁺ in Myo-GFP⁺ cells was quantified. A significant increase in EC death was observed in the gut following EC-specific VDR knockdown (Figure 1B). These results indicate that VDR is required for EC survival under normal conditions.

Figure 2. Effect of EC-specific VDR knockdown on EC cell death. (A) EC-specific knockdown of VDR induces cleaved caspase-3 in ECs. Flies carrying the Myo^ts>GFP or Myo^ts>GFP+VDRRi genotypes were cultured at 29° C for 4 days. The guts of flies were dissected and labeled with anti-GFP (green) and anti-cleaved caspase-3 (red) antibodies and DAPI (blue). Original magnification is 400×. (B) Frequency of cleaved-caspase 3⁺ cells per Myo⁺ (strong and weak GFP⁺) large cell (ECs). Three-day-old females were shifted to 29° C for 4 days, and dissected guts were immunostained with anti-GFP (green) and anti-cleaved caspase 3 (red) antibodies and DAPI (blue). Cleaved caspase 3⁺ cell numbers were determined in the Myo⁺ cells of these guts. Data (mean ± standard error) in the Myo^ts>GFP or Myo^ts>GFP+VDRRi flies were collated from 4716 and 5914 Myo⁺ cells of 15 guts each. P-values were determined using Student’s t-test. p<0.0001 compared with Myo^ts>GFP flies.

VDR knockdown in ECs induces ISC aging

To assess the implications of excessive ISC proliferation due to EC-specific VDR knockdown-induced EC death, damage accumulation in ISCs was analyzed using antibodies against γH2AvD (a molecular marker of DNA double strand breaks) [14, 45], and Pros (an EE marker). The γH2AvD signal was exceedingly low in the Myo-GFP^- and Pros^- cells (ISCs) of Myo^ts>GFP flies (Figure 3A, left panel); however, γH2AvD foci were markedly increased in the Myo-GFP^- and Pros^- ISCs of Myo^ts>GFP+VDRRi flies (Figure 3A, right panel). This indicates that the EC-specific knockdown of VDR could induce DNA damage accumulation in ISCs. Centrosome amplification (a hallmark of cancer cells) was also examined using anti-γ-tubulin and anti-PH3 antibodies. In control files, two centrosomes in mitotic ISCs (PH3⁺ cells) were detected; however, mitotic ISCs with 3–12 abnormal centrosomes were detected in the EC-specific VDR-knockdown flies carrying the Myo^ts>GFP+VDRRi genotype (Figure 3B a). Subsequently, the number of PH3⁺ cells per gut was quantified, and 114.05 were observed in Myo^ts>GFP+VDRRi flies (N = 20, n = 2281), while 7.33 were observed in Myo^ts>GFP flies (N = 21, n = 154) (Figure 3B b). In addition, the frequencies of these mitotic ISCs with supernumerary centrosomes (>2) were quantified, revealing values of 8.03 and 0.35% in Myo^ts>GFP+VDRRi and Myo^ts>GFP flies, respectively (Figure 3B c). The number of mitotic ISCs with supernumerary centrosomes (>2) per gut was 9.25 in the Myo^ts>GFP+ VDRRi flies and 0.09 in the Myo^ts>GFP flies (Figure 3B d). These results show that VDR inhibition resulted in DNA damage accumulation and a higher incidence of centrosome amplification in ISCs.

Figure 3. EC-specific VDR knockdown increases age-related ISC phenotypes. (A) EC-specific VDR knockdown induces DNA damage accumulation in ISCs and their progenitors. Flies carrying the Myo^ts>GFP or Myo^ts>GFP+VDRRi genotype were incubated at 29° C for 7 days. The guts of flies were dissected and labeled with anti-γH2AvD (red), anti-Pros (white), and anti-GFP (green) antibodies and DAPI (blue). (B) EC-specific VDR knockdown causes centrosome amplification in mitotic ISCs. Flies carrying the Myo^ts>GFP or Myo^ts>GFP+VDRRi genotype were incubated at 29° C for 7 days. (a) The entire guts of the flies were dissected, fixed, and labeled with anti-γ-tubulin (red), anti-PH3 (white), and anti-GFP (green) antibodies and DAPI (blue). The original magnification is 400×. (b–d) The number of mitotic ISCs with supernumerary centrosomes (>2) in the midguts of Myo^ts>GFP or Myo^ts>GFP+VDRRi flies increased. (b) EC-specific VDR knockdown increases mitotic ISCs in the guts. (c) Frequency of cells with supernumerary centrosomes per mitotic ISC. (d) Number of cells with supernumerary centrosomes per midgut. Three-day-old female flies were cultured at 29° C for 7 days, and then their dissected guts were fixed and immunostained with anti-γ-tubulin (red), anti-PH3 (white), and anti-GFP (green) antibodies and DAPI (blue). In these guts, the number of abnormal centrosomes in the PH3⁺ cells were determined. Data (mean ± SD) in Myo^ts>GFP or Myo^ts>GFP+VDRRi flies were collated from 154 and 2281 mitotic cells of 21 and 20 guts, respectively. P-values were calculated using Student’s t-test. P < 0.0001 compared with Myo^ts>GFP flies.

VDR knockdown in ECs induces EE differentiation

Following EC-specific VDR knockdown, the increase in Dl⁺ cell numbers in the gut was analyzed using an anti-Dl antibody (Dl is a molecular marker of ISCs). Unlike the previously reported increase in the number of strong Dl⁺ cells (ISC-EB-EC) following the age-related increase in ISC proliferation [8–10], very weak Dl⁺ cells and a marked decrease in Dl⁺ cell numbers in guts with EC-specific VDR knockdown were observed (Figure 4A, 4B). Therefore, to evaluate ISC differentiation in guts with EC-specific VDR knockdown, the number of Pros⁺ cells in these guts was analyzed using an anti-Pros antibody since Pros is a molecular marker of EE. The number of EEs significantly increased in the guts of Myo^ts>GFP+VDRRi flies compared with the guts of wild-type Myo^ts>GFP flies (Figure 5A and Supplementary Figure 1). Furthermore, the EE ratio in the midgut was quantified. Significant increases in EEs were detected in the guts of EC-specific VDR knockdown flies (Figure 5B and Supplementary Figure 1). This indicates that VDR knockdown in ECs induces an excessive increase in EE numbers. Additionally, we evaluated whether VDR knockdown-induced EE differentiation was ameliorated by VitD treatment. First, to this end, it was determined that VitD could activate the VDR pathway by feeding adult flies with VitD using an anti-hVDR antibody. That is, 7-day-old wild-type flies (Oregon-R) were fed with 100 nM 1α,25-Dihydroxyvitamin D₃ for 24 h. In wild-type flies not fed 1α,25-Dihydroxyvitamin D₃, VDRs were scattered throughout the intestinal cells, including ECs and EE cells (Supplementary Figure 2, left panels). In contrast, VDRs migrated to the nuclear membrane in the intestinal cells of flies fed 1α,25-Dihydroxyvitamin D₃ (Supplementary Figure 2, right panels). Second, we determined whether VitD could activate the VDR pathway in Myo^ts>GFP and Myo^ts>GFP+hr96Ri flies (Supplementary Figure 3). As hypothesized, EC-specific VDR knockdown-induced EE differentiation was ameliorated by VitD treatment (Supplementary Figure 4). These results indicated that the VitD/VDR (Hr96) pathway in fly intestinal models functions similarly to that in mammals. Therefore, VDR is involved in EE differentiation under normal conditions.

Figure 4. EC-specific VDR knockdown decreases the number of Dl⁺ cells. (A) EC-specific knockdown of VDR decreases the number of Delta⁺ cells. Flies carrying the Myo^ts>GFP or Myo^ts>GFP+VDRRi genotypes were incubated at 29° C for 1 week. The whole guts of flies were dissected, fixed, and labeled with anti-Dl (red) and anti-GFP (green) antibodies and DAPI (blue). The original magnification is 400×. (B) Frequency of Dl⁺ cells per Myo^- small cell. Three-day-old females were shifted to 29° C for 1 week, and their dissected guts were fixed and immunostained with anti-Dl (red) and anti-GFP (green) antibodies and DAPI (blue). The number of Dl⁺ cells in the Myo^- small cells was determined in these guts. Data (mean ± standard error) in Myo^ts>GFP or Myo^ts>GFP+VDRRi flies were collated from 882 and 1192 Myo^- cells of 16 and 15 guts, respectively. P-values were determined using Student’s t-test. P < 0.0001 compared with Myo^ts>GFP flies.

Figure 5. EC-specific VDR knockdown increases the number of Pros⁺ cells. (A) EC-specific VDR knockdown increases Pros⁺ cell number. Flies carrying the Myo^ts>GFP or Myo^ts>GFP+VDRRi genotypes were incubated at 29° C for 1 week. The whole guts of flies were dissected, fixed, and labeled with anti-Pros (red) and anti-GFP (green) antibodies and DAPI (blue). The original magnification is 200×. (B) Frequency of Pros⁺ cells per total cells. Three-day-old females were shifted to 29° C for 1 week, and their dissected guts were fixed and immunostained with anti-Pros (red) and anti-GFP (green) antibodies and DAPI (blue). The Pros⁺ cell numbers were recorded with respect to the total cells of these guts. Data (mean ± standard error) in Myo^ts>GFP or Myo^ts>GFP+VDRRi flies were collated from 7459 and 7992 total cells of 19 guts each, respectively. P-values were determined using Student’s t-test. P<0.0001 compared with Myo^ts>GFP flies.

Inhibitory effect of VitD on age- and oxidative stress-related ISC proliferation and centrosome amplification in the midgut

The effect of VitD on age-related phenotypes (increased cell proliferation and centrosome amplification) in ISCs was investigated. To investigate the anti-aging effect of VitD, ISCs with or without 100 nM 1α,25-Dihydroxyvitamin D₃ treatment were stained with antibodies against γ-tubulin (a centrosome marker), PH3 (a mitotic ISCs marker), and GFP (an ECs marker), and the number of cells displaying centrosome amplification was assessed. Supernumerary centrosomes were observed in 4.64% of mitotic ISCs in 45-day-old Myo^ts>GFP flies (Figure 6A, 6C) and in 6.7% of 10-day-old Myo^ts>GFP+Catⁿ¹ flies (Figure 6A, 6C), a model of intrinsic oxidative stress [8], compared with 0.35% in 10-day-old Myo^ts>GFP flies (Figure 6A, 6C). The number of mitotic ISCs with supernumerary centrosomes per gut was 2.59 in 45-day-old Myo^ts>GFP flies and 7.81 in 10-day-old Myo^ts>GFP+Catⁿ¹ flies compared with 0.08 in 10-day-old Myo^ts>GFP flies (Figure 6D). The age- and oxidative stress-related increase in the number of PH3⁺ cells was reduced by VitD treatment (Figure 6B). In addition, VitD treatment reduced the age- and oxidative stress-related increase in supernumerary centrosomes in 2.45 and 4.86% of mitotic ISCs in 45-day-old Myo^ts>GFP and 10-day-old Myo^ts>GFP+Catⁿ¹ flies, respectively (Figure 6A, 6C). The number of mitotic ISCs with supernumerary centrosomes per gut was reduced by 0.94 in 45-day-old Myo^ts>GFP flies, and by 3.67 in 10-day-old Myo^ts>GFP+Catⁿ¹ flies, whereas no change was observed in 10-day-old Myo^ts>GFP flies (Figure 6A, 6C). Additionally, to determine whether the inhibitory effect of VitD on age-related phenotypes is associated with the VDR pathway, the VDR in ECs was knocked down. For EC-specific expression, 3-day-old Myo^ts>GFP and Myo^ts>GFP+hr96Ri flies were cultured at 29° C for 7 days. Compared with the untreated group, VitD treatment reduced mitotic ISCs and the number of mitotic ISCs with supernumerary centrosomes in the guts of Myo^ts>GFP flies in which VDR was knocked down (Figure 6A, 6B). In contrast, VitD treatment did not affect the intestinal morphology of 10-day-old Myo^ts>GFP flies. These results indicate that VitD can reduce age- and oxidative stress-induced centrosome amplification in adult ISCs in vivo.

Figure 6. Inhibitory effect of VitD on age- and oxidative stress-related accumulation of supernumerary centrosomes in midgut ISCs. (A) The guts from 10-day-old Myo^ts>GFP flies, 45-day-old Myo^ts>GFP flies, 10-day-old Myo^ts>GFP+Catⁿ¹ mutant flies, and 10-day-old Myo^ts>GFP+hr96Ri flies, without or with 100 nM VitD feeding for 1 week were stained with anti-PH3 (white), anti-γ-tubulin (red), anti-GFP (green), and DAPI (blue). The original magnification is 400×. (B) The number of PH3⁺ cells in whole guts from 10-day-old Myo^ts>GFP, 45-day-old Myo^ts>GFP, 10-day-old Myo^ts>GFP+Catⁿ¹, and Myo^ts>GFP+hr96Ri flies, with or without VitD feeding for 7 days was determined. Data (mean ± standard error) in 10-day-old Myo^ts>GFP, 45-day-old Myo^ts>GFP, 10-day-old Myo^ts>GFP+Catⁿ¹, and Myo^ts>GFP+hr96Ri flies without VitD feeding were collated from 165, 823, 1835, and 2484 mitotic cells of 25, 17, 16, and 22 guts, respectively. Data (mean ± standard error) in 10-day-old Myo^ts>GFP, 45-day-old Myo^ts>GFP, 10-day-old Myo^ts>GFP+Catⁿ¹, and Myo^ts>GFP+hr96Ri flies with VitD feeding were collated from 126, 489, 1235, and 1445 mitotic cells of 28, 18, 18, and 19 guts, respectively. n.s., no significant difference from the control (p > 0.05). (C) The frequency of supernumerary centrosomes (>2) per mitotic ISC in 10-day-old Myo^ts>GFP, 45-day-old Myo^ts>GFP, 10-day-old Myo^ts>GFP+Catⁿ¹, and Myo^ts>GFP+hr96Ri flies with or without VitD feeding for 7 days. The centrosome numbers in mitotic ISCs (PH3⁺ and GFP^- cells) in the midgut were determined. n.s., no significant difference from the control (p>0.05). (D) The frequency of mitotic ISCs with supernumerary centrosomes per gut in 10-day-old Myo^ts>GFP, 45-day-old Myo^ts>GFP, 10-day-old Myo^ts>GFP+Catⁿ¹, and 10-day-old Myo^ts>GFP+hr96Ri flies with or without VitD feeding for 1 week. Error bars represent standard error. P-values were calculated using Student’s t-test. n.s., no significant difference from the control (p>0.05).

Abbreviations

ISC: intestinal stem cell; EB: enteroblast; EC: enterocyte; EE: enteroendocrine; VDR: vitamin D receptor; GFP: green fluorescent protein; DAPI: 4’,6-diamidino-2-phenylindole; SD: standard deviation; γH2AvD: phosphorylated Drosophila histone variant of H2A (Ser137); EGFR: epidermal growth factor receptor; JNK: c-Jun N-terminal kinase; PH3: phosphorylated histone H3; HP1: heterochromatin protein 1; TOR: target of rapamycin; PVR: PDGF- and VEGF-receptor related; ATM/ATR: ataxia telangiectasia-mutated (ATM) and ATM- and RAD3-related (ATR).

Author Contributions

J.S.P. conceived the study, designed the methods and experiments, performed the experiments, analyzed the data, interpreted the results, and wrote the manuscript. H.J.N. designed the experiment and discussed the manuscript. Y.J.K. discussed the manuscript. All authors have read and agreed to the published version of the manuscript.

Acknowledgments

We would like to thank Prof. M.A. Yoo (Pusan National University, Busan, Republic of Korea) for invaluable comments regarding the manuscript, Prof. B.A. Edgar for the fly stocks (Myo^ts>GFP), and the DSHB for the antibodies. We also thank the BDSC, VDRC, and DGRC for the Drosophila stocks.

Conflicts of Interest

The authors of this manuscript declare no conflicts of interest.

Funding

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2019R1A2C1005302, RS-2023-00243729).

References

• 1. Rando TA. Stem cells, ageing and the quest for immortality. Nature. 2006; 441:1080–6. https://doi.org/10.1038/nature04958 [PubMed]
• 2. Spradling A, Drummond-Barbosa D, Kai T. Stem cells find their niche. Nature. 2001; 414:98–104. https://doi.org/10.1038/35102160 [PubMed]
• 3. Micchelli CA, Perrimon N. Evidence that stem cells reside in the adult Drosophila midgut epithelium. Nature. 2006; 439:475–9. https://doi.org/10.1038/nature04371 [PubMed]
• 4. Ohlstein B, Spradling A. The adult Drosophila posterior midgut is maintained by pluripotent stem cells. Nature. 2006; 439:470–4. https://doi.org/10.1038/nature04333 [PubMed]
• 5. Ohlstein B, Spradling A. Multipotent Drosophila intestinal stem cells specify daughter cell fates by differential notch signaling. Science. 2007; 315:988–92. https://doi.org/10.1126/science.1136606 [PubMed]
• 6. Sahai-Hernandez P, Castanieto A, Nystul TG. Drosophila models of epithelial stem cells and their niches. Wiley Interdiscip Rev Dev Biol. 2012; 1:447–57. https://doi.org/10.1002/wdev.36 [PubMed]
• 7. Lee WC, Beebe K, Sudmeier L, Micchelli CA. Adenomatous polyposis coli regulates Drosophila intestinal stem cell proliferation. Development. 2009; 136:2255–64. https://doi.org/10.1242/dev.035196 [PubMed]
• 8. Choi NH, Kim JG, Yang DJ, Kim YS, Yoo MA. Age-related changes in Drosophila midgut are associated with PVF2, a PDGF/VEGF-like growth factor. Aging Cell. 2008; 7:318–34. https://doi.org/10.1111/j.1474-9726.2008.00380.x [PubMed]
• 9. Biteau B, Hochmuth CE, Jasper H. JNK activity in somatic stem cells causes loss of tissue homeostasis in the aging Drosophila gut. Cell Stem Cell. 2008; 3:442–55. https://doi.org/10.1016/j.stem.2008.07.024 [PubMed]
• 10. Park JS, Kim YS, Yoo MA. The role of p38b MAPK in age-related modulation of intestinal stem cell proliferation and differentiation in Drosophila. Aging (Albany NY). 2009; 1:637–51. https://doi.org/10.18632/aging.100054 [PubMed]
• 11. Buchon N, Broderick NA, Poidevin M, Pradervand S, Lemaitre B. Drosophila intestinal response to bacterial infection: activation of host defense and stem cell proliferation. Cell Host Microbe. 2009; 5:200–11. https://doi.org/10.1016/j.chom.2009.01.003 [PubMed]
• 12. Buchon N, Broderick NA, Chakrabarti S, Lemaitre B. Invasive and indigenous microbiota impact intestinal stem cell activity through multiple pathways in Drosophila. Genes Dev. 2009; 23:2333–44. https://doi.org/10.1101/gad.1827009 [PubMed]
• 13. Lee WJ. Bacterial-modulated host immunity and stem cell activation for gut homeostasis. Genes Dev. 2009; 23:2260–5. https://doi.org/10.1101/gad.1858709 [PubMed]
• 14. Park JS, Lee SH, Na HJ, Pyo JH, Kim YS, Yoo MA. Age- and oxidative stress-induced DNA damage in Drosophila intestinal stem cells as marked by Gamma-H2AX. Exp Gerontol. 2012; 47:401–5. https://doi.org/10.1016/j.exger.2012.02.007 [PubMed]
• 15. Na HJ, Park JS, Pyo JH, Lee SH, Jeon HJ, Kim YS, Yoo MA. Mechanism of metformin: inhibition of DNA damage and proliferative activity in Drosophila midgut stem cell. Mech Ageing Dev. 2013; 134:381–90. https://doi.org/10.1016/j.mad.2013.07.003 [PubMed]
• 16. Park JS, Pyo JH, Na HJ, Jeon HJ, Kim YS, Arking R, Yoo MA. Increased centrosome amplification in aged stem cells of the Drosophila midgut. Biochem Biophys Res Commun. 2014; 450:961–5. https://doi.org/10.1016/j.bbrc.2014.06.085 [PubMed]
• 17. Beebe K, Lee WC, Micchelli CA. JAK/STAT signaling coordinates stem cell proliferation and multilineage differentiation in the Drosophila intestinal stem cell lineage. Dev Biol. 2010; 338:28–37. https://doi.org/10.1016/j.ydbio.2009.10.045 [PubMed]
• 18. Jiang H, Grenley MO, Bravo MJ, Blumhagen RZ, Edgar BA. EGFR/Ras/MAPK signaling mediates adult midgut epithelial homeostasis and regeneration in Drosophila. Cell Stem Cell. 2011; 8:84–95. https://doi.org/10.1016/j.stem.2010.11.026 [PubMed]
• 19. Bond D, Foley E. Autocrine platelet-derived growth factor-vascular endothelial growth factor receptor-related (Pvr) pathway activity controls intestinal stem cell proliferation in the adult Drosophila midgut. J Biol Chem. 2012; 287:27359–70. https://doi.org/10.1074/jbc.M112.378018 [PubMed]
• 20. Ren F, Wang B, Yue T, Yun EY, Ip YT, Jiang J. Hippo signaling regulates Drosophila intestine stem cell proliferation through multiple pathways. Proc Natl Acad Sci USA. 2010; 107:21064–9. https://doi.org/10.1073/pnas.1012759107 [PubMed]
• 21. Shaw RL, Kohlmaier A, Polesello C, Veelken C, Edgar BA, Tapon N. The Hippo pathway regulates intestinal stem cell proliferation during Drosophila adult midgut regeneration. Development. 2010; 137:4147–58. https://doi.org/10.1242/dev.052506 [PubMed]
• 22. Karpowicz P, Perez J, Perrimon N. The Hippo tumor suppressor pathway regulates intestinal stem cell regeneration. Development. 2010; 137:4135–45. https://doi.org/10.1242/dev.060483 [PubMed]
• 23. Choi NH, Lucchetta E, Ohlstein B. Nonautonomous regulation of Drosophila midgut stem cell proliferation by the insulin-signaling pathway. Proc Natl Acad Sci USA. 2011; 108:18702–7. https://doi.org/10.1073/pnas.1109348108 [PubMed]
• 24. Na HJ, Park JS, Pyo JH, Jeon HJ, Kim YS, Arking R, Yoo MA. Metformin inhibits age-related centrosome amplification in Drosophila midgut stem cells through AKT/TOR pathway. Mech Ageing Dev. 2015; 149:8–18. https://doi.org/10.1016/j.mad.2015.05.004 [PubMed]
• 25. Fan X, Liang Q, Lian T, Wu Q, Gaur U, Li D, Yang D, Mao X, Jin Z, Li Y, Yang M. Rapamycin preserves gut homeostasis during Drosophila aging. Oncotarget. 2015; 6:35274–83. https://doi.org/10.18632/oncotarget.5895 [PubMed]
• 26. Zhou J, Florescu S, Boettcher AL, Luo L, Dutta D, Kerr G, Cai Y, Edgar BA, Boutros M. Dpp/Gbb signaling is required for normal intestinal regeneration during infection. Dev Biol. 2015; 399:189–203. https://doi.org/10.1016/j.ydbio.2014.12.017 [PubMed]
• 27. Osman D, Buchon N, Chakrabarti S, Huang YT, Su WC, Poidevin M, Tsai YC, Lemaitre B. Autocrine and paracrine unpaired signaling regulate intestinal stem cell maintenance and division. J Cell Sci. 2012; 125:5944–9. https://doi.org/10.1242/jcs.113100 [PubMed]
• 28. Li VS, Clevers H. Intestinal regeneration: YAP-tumor suppressor and oncoprotein? Curr Biol. 2013; 23:R110–2. https://doi.org/10.1016/j.cub.2012.12.021 [PubMed]
• 29. Lin G, Xu N, Xi R. Paracrine Wingless signalling controls self-renewal of Drosophila intestinal stem cells. Nature. 2008; 455:1119–23. https://doi.org/10.1038/nature07329 [PubMed]
• 30. Biteau B, Jasper H. EGF signaling regulates the proliferation of intestinal stem cells in Drosophila. Development. 2011; 138:1045–55. https://doi.org/10.1242/dev.056671 [PubMed]
• 31. O’Brien LE, Soliman SS, Li X, Bilder D. Altered modes of stem cell division drive adaptive intestinal growth. Cell. 2011; 147:603–14. https://doi.org/10.1016/j.cell.2011.08.048 [PubMed]
• 32. Li Z, Zhang Y, Han L, Shi L, Lin X. Trachea-derived dpp controls adult midgut homeostasis in Drosophila. Dev Cell. 2013; 24:133–43. https://doi.org/10.1016/j.devcel.2012.12.010 [PubMed]
• 33. Park JS, Na HJ, Pyo JH, Jeon HJ, Kim YS, Yoo MA. Requirement of ATR for maintenance of intestinal stem cells in aging Drosophila. Aging (Albany NY). 2015; 7:307–18. https://doi.org/10.18632/aging.100743 [PubMed]
• 34. Randerson-Moor JA, Taylor JC, Elliott F, Chang YM, Beswick S, Kukalizch K, Affleck P, Leake S, Haynes S, Karpavicius B, Marsden J, Gerry E, Bale L, et al. Vitamin D receptor gene polymorphisms, serum 25-hydroxyvitamin D levels, and melanoma: UK case-control comparisons and a meta-analysis of published VDR data. Eur J Cancer. 2009; 45:3271–81. https://doi.org/10.1016/j.ejca.2009.06.011 [PubMed]
• 35. Caini S, Boniol M, Tosti G, Magi S, Medri M, Stanganelli I, Palli D, Assedi M, Marmol VD, Gandini S. Vitamin D and melanoma and non-melanoma skin cancer risk and prognosis: a comprehensive review and meta-analysis. Eur J Cancer. 2014; 50:2649–58. https://doi.org/10.1016/j.ejca.2014.06.024 [PubMed]
• 36. Berridge MJ. Vitamin D cell signalling in health and disease. Biochem Biophys Res Commun. 2015; 460:53–71. https://doi.org/10.1016/j.bbrc.2015.01.008 [PubMed]
• 37. Warwick T, Schulz MH, Günther S, Gilsbach R, Neme A, Carlberg C, Brandes RP, Seuter S. A hierarchical regulatory network analysis of the vitamin D induced transcriptome reveals novel regulators and complete VDR dependency in monocytes. Sci Rep. 2021; 11:6518. https://doi.org/10.1038/s41598-021-86032-5 [PubMed]
• 38. Fisk GJ, Thummel CS. Isolation, regulation, and DNA-binding properties of three Drosophila nuclear hormone receptor superfamily members. Proc Natl Acad Sci USA. 1995; 92:10604–8. https://doi.org/10.1073/pnas.92.23.10604 [PubMed]
• 39. Wilk R, Hu J, Krause HM. Spatial profiling of nuclear receptor transcription patterns over the course of Drosophila development. G3 (Bethesda). 2013; 3:1177–89. https://doi.org/10.1534/g3.113.006023 [PubMed]
• 40. McKenna NJ, O’Malley BW. Combinatorial control of gene expression by nuclear receptors and coregulators. Cell. 2002; 108:465–74. https://doi.org/10.1016/s0092-8674(02)00641-4 [PubMed]
• 41. King-Jones K, Horner MA, Lam G, Thummel CS. The DHR96 nuclear receptor regulates xenobiotic responses in Drosophila. Cell Metab. 2006; 4:37–48. https://doi.org/10.1016/j.cmet.2006.06.006 [PubMed]
• 42. Horner MA, Pardee K, Liu S, King-Jones K, Lajoie G, Edwards A, Krause HM, Thummel CS. The Drosophila DHR96 nuclear receptor binds cholesterol and regulates cholesterol homeostasis. Genes Dev. 2009; 23:2711–6. https://doi.org/10.1101/gad.1833609 [PubMed]
• 43. Sieber MH, Thummel CS. The DHR96 nuclear receptor controls triacylglycerol homeostasis in Drosophila. Cell Metab. 2009; 10:481–90. https://doi.org/10.1016/j.cmet.2009.10.010 [PubMed]
• 44. Obniski R, Sieber M, Spradling AC. Dietary Lipids Modulate Notch Signaling and Influence Adult Intestinal Development and Metabolism in Drosophila. Dev Cell. 2018; 47:98–111.e5. https://doi.org/10.1016/j.devcel.2018.08.013 [PubMed]
• 45. Jeon HJ, Kim YS, Park JS, Pyo JH, Na HJ, Kim IJ, Kim CM, Chung HY, Kim ND, Arking R, Yoo MA. Age-related change in γH2AX of Drosophila muscle: its significance as a marker for muscle damage and longevity. Biogerontology. 2015; 16:503–16. https://doi.org/10.1007/s10522-015-9573-0 [PubMed]
• 46. Amcheslavsky A, Jiang J, Ip YT. Tissue damage-induced intestinal stem cell division in Drosophila. Cell Stem Cell. 2009; 4:49–61. https://doi.org/10.1016/j.stem.2008.10.016 [PubMed]
• 47. Park JS, Jeon HJ, Pyo JH, Kim YS, Yoo MA. Deficiency in DNA damage response of enterocytes accelerates intestinal stem cell aging in Drosophila. Aging (Albany NY). 2018; 10:322–38. https://doi.org/10.18632/aging.101390 [PubMed]
• 48. Halicka HD, Zhao H, Li J, Traganos F, Studzinski GP, Darzynkiewicz Z. Attenuation of constitutive DNA damage signaling by 1,25-dihydroxyvitamin D3. Aging (Albany NY). 2012; 4:270–8. https://doi.org/10.18632/aging.100450 [PubMed]
• 49. Erez N, Israitel L, Bitman-Lotan E, Wong WH, Raz G, Cornelio-Parra DV, Danial S, Flint Brodsly N, Belova E, Maksimenko O, Georgiev P, Druley T, Mohan RD, Orian A. A Non-stop identity complex (NIC) supervises enterocyte identity and protects from premature aging. Elife. 2021; 10:e62312. https://doi.org/10.7554/eLife.62312 [PubMed]
• 50. Lu YX, Regan JC, Eßer J, Drews LF, Weinseis T, Stinn J, Hahn O, Miller RA, Grönke S, Partridge L. A TORC1-histone axis regulates chromatin organisation and non-canonical induction of autophagy to ameliorate ageing. Elife. 2021; 10:e62233. https://doi.org/10.7554/eLife.62233 [PubMed]
• 51. Nan Y, Lin J, Cui Y, Yao J, Yang Y, Li Q. Protective role of vitamin B6 against mitochondria damage in Drosophila models of SCA3. Neurochem Int. 2021; 144:104979. https://doi.org/10.1016/j.neuint.2021.104979 [PubMed]
• 52. Khayami R, Goltzman D, Rabbani SA, Kerachian MA. Epigenomic effects of vitamin D in colorectal cancer. Epigenomics. 2022; 14:1213–28. https://doi.org/10.2217/epi-2022-0288 [PubMed]
• 53. Jiang H, Patel PH, Kohlmaier A, Grenley MO, McEwen DG, Edgar BA. Cytokine/Jak/Stat signaling mediates regeneration and homeostasis in the Drosophila midgut. Cell. 2009; 137:1343–55. https://doi.org/10.1016/j.cell.2009.05.014 [PubMed]
• 54. Biteau B, Jasper H. Slit/Robo signaling regulates cell fate decisions in the intestinal stem cell lineage of Drosophila. Cell Rep. 2014; 7:1867–75. https://doi.org/10.1016/j.celrep.2014.05.024 [PubMed]
• 55. Lind A, Wängberg B, Ellegård L. Vitamin D and vitamin B12 deficiencies are common in patients with midgut carcinoid (SI-NET). Eur J Clin Nutr. 2016; 70:990–4. https://doi.org/10.1038/ejcn.2016.40 [PubMed]
• 56. Fagan R, Bokhari SSN, Inayat F. Vitamin D and vitamin B₁₂ deficiencies in patients with small intestinal carcinoid tumour: is opioid use disorder a confounding factor in the diagnosis? BMJ Case Rep. 2019; 12:e227430. https://doi.org/10.1136/bcr-2018-227430 [PubMed]
• 57. Altieri B, Barrea L, Modica R, Bottiglieri F, de Cicco F, Muscogiuri G, Circelli L, Savarese G, Di Somma C, Savastano S, Colao A, Faggiano A. Vitamin D deficiency and tumor aggressiveness in gastroenteropancreatic neuroendocrine tumors. Endocrine. 2022; 75:623–34. https://doi.org/10.1007/s12020-021-02869-w [PubMed]
• 58. Fang Y, Song H, Huang J, Zhou J, Ding X. The clinical significance of vitamin D levels and vitamin D receptor mRNA expression in colorectal neoplasms. J Clin Lab Anal. 2021; 35:e23988. https://doi.org/10.1002/jcla.23988 [PubMed]
• 59. Park JS, Kim YJ. Anti-Aging Effect of the Ketone Metabolite β-Hydroxybutyrate in Drosophila Intestinal Stem Cells. Int J Mol Sci. 2020; 21:3497. https://doi.org/10.3390/ijms21103497 [PubMed]
• 60. Mitsuo T, Nakao M. [Vitamin D and anti-aging medicine]. Clin Calcium. 2008; 18:980–5. [PubMed]
• 61. Li W, Che X, Chen X, Zhou M, Luo X, Liu T. Study of calcitriol anti-aging effects on human natural killer cells in vitro. Bioengineered. 2021; 12:6844–54. https://doi.org/10.1080/21655979.2021.1972076 [PubMed]
• 62. Sosa-Díaz E, Hernández-Cruz EY, Pedraza-Chaverri J. The role of vitamin D on redox regulation and cellular senescence. Free Radic Biol Med. 2022; 193:253–73. https://doi.org/10.1016/j.freeradbiomed.2022.10.003 [PubMed]
• 63. Giustina A, Bouillon R, Dawson-Hughes B, Ebeling PR, Lazaretti-Castro M, Lips P, Marcocci C, Bilezikian JP. Vitamin D in the older population: a consensus statement. Endocrine. 2023; 79:31–44. https://doi.org/10.1007/s12020-022-03208-3 [PubMed]
• 64. Hamada T, Liu L, Nowak JA, Mima K, Cao Y, Ng K, Twombly TS, Song M, Jung S, Dou R, Masugi Y, Kosumi K, Shi Y, et al. Vitamin D status after colorectal cancer diagnosis and patient survival according to immune response to tumour. Eur J Cancer. 2018; 103:98–107. https://doi.org/10.1016/j.ejca.2018.07.130 [PubMed]
• 65. Griswold CM, Matthews AL, Bewley KE, Mahaffey JW. Molecular characterization and rescue of acatalasemic mutants of Drosophila melanogaster. Genetics. 1993; 134:781–8. https://doi.org/10.1093/genetics/134.3.781 [PubMed]
• 66. McGuire SE, Roman G, Davis RL. Gene expression systems in Drosophila: a synthesis of time and space. Trends Genet. 2004; 20:384–91. https://doi.org/10.1016/j.tig.2004.06.012 [PubMed]