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. 2021 Sep 25;12(10):874.
doi: 10.1038/s41419-021-04147-z.

Fear-of-intimacy-mediated zinc transport controls fat body cell dissociation through modulating Mmp activity in Drosophila

Tian Wei #  1 , Xiaowen Ji #  1 , Qunhui Yu #  1 , Guangying Li  1 , Lei Wu  1 , Yan Gao  1 , Guiran Xiao  2
Affiliations

Fear-of-intimacy-mediated zinc transport controls fat body cell dissociation through modulating Mmp activity in Drosophila

Tian Wei et al. Cell Death Dis. .

Abstract

Matrix metalloproteinases (Mmps) are pivotal extracellular proteinases that have been implicated in tumour invasion and metastasis. Drosophila fat body is important for energy storage and utilization, as well as biosynthetic and metabolic activities. The fat body undergoes remodelling during metamorphosis which is characterized by the dissociation of the fat body into individual cells. Mmps play important roles in the regulation of fat body cell dissociation. Here we show that a zinc transporter fear-of-intimacy (foi) is necessary for the cell dissociation of fat body in Drosophila. The progression of fat body cell dissociation was delayed by fat body-specific foi knockdown while it was accelerated by foi overexpression (OE). In essence, these phenotypes are closely associated with intracellular zinc homeostasis, which can be modulated by dietary zinc intervention or genetic modulation of other zinc transporters. Further study indicated that Mmp1 and Mmp2 levels could be transcriptionally regulated by zinc in vivo. Consistently, the retarded fat body cell dissociation caused by Mmp1 or Mmp2 RNAi could be regulated by modulating the expression of foi. Further, by using Drosophila models of malignant tumour RafGOFscrib-/- and RasV12lgl-/-, we showed that the tumour growth, invasion and migration could be markedly inhibited by foi knockdown. These findings demonstrate a close connection between zinc levels and cell dissociation in vivo, and also suggest that manipulation of zinc levels may provide a novel therapeutic strategy for cancer.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Foi maintains zinc homeostasis in the fat body and modulates the development of Drosophila melanogaster.
A Fat body-specific foi knockdown (Cg-Gal4 > foi RNAi) resulted in dramatically reduced eclosion rate in Drosophila. n = 70 larvae per vial, n = 6 vials per group. B The defects of RNA-interference foi (Cg-Gal4 > foi RNAi) flies could be absolutely rescued by dietary zinc supplementation (2 mM ZnCl2) while aggravated by zinc chelator (25 mM TPEN). C The Zinpyr-1 staining revealed that fat body-specific foi OE resulted in zinc accumulation while foi RNAi decreased zinc particles in the fat body cells. D The activity of ALP was significantly reduced in Lsp2-Gal4 > foi RNAi larvae while increased in Lsp2-Gal4 > foi OE larvae. w1118 flies driven by Cg-Gal4 or Lsp2-Gal4 serve as a blank control. All the experiments were conducted independently at least for 3 times. ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed Student’s t test.
Fig. 2
Fig. 2. Cytosolic zinc affects the progression of the fat body cell dissociation.
A, B The fat body cell dissociation was greatly delayed in Lsp2-Gal4 > foi RNAi while accelerated in Lsp2-Gal4 > foi OE. C, D Dietary zinc supplementation was able to rescue the retarded fat body cell dissociation induced by fat body-specific foi RNAi, while enhance the precocious the progression induced by foi OE. E, F Zinc chelator TPEN had the capability of rescuing the precocious fat body cell dissociation induced by foi OE, while aggravating the retarded fat body cell dissociation induced by foi RNAi. G, H ZnT1 OE in the fat body dramatically rescued the precocious cell dissociation caused by fat body-specific foi OE, while enhanced the retarded cell dissociation induced by foi RNAi. All the experiments were conducted independently at least for 3 times. ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed Student’s t test.
Fig. 3
Fig. 3. Foi is required for the structural stabilization of fat body cells.
The fat body structure was investigated with four hallmarks at the wandering stage, 4 h APF and 7 h APF. A Phalloidin staining revealed the shape of the fat body cells. B, C Immunostaining of DE-cadherin (B) and integrin βPs (C) were used to monitor the cell–cell junctions and cell–basement membrane (BM) junctions, respectively. D Viking-GFP revealed the integrity of the BM. A nearly intact BM was observed in the fat body of Lsp2-Gal4 > foi RNAi animals, while the BM was broken in Lsp2-Gal4 > foi OE animals. w1118 flies driven by Lsp2-Gal4 serve as a blank control. The arrows in (A–C) indicated the gaps between cells, and the arrows in (D) showed the degraded BM.
Fig. 4
Fig. 4. Zinc affects the enzyme activity, protein and mRNA levels of the two Mmps.
A Gelatin zymogram revealed that total Mmp enzyme activity was considerably diminished in Lsp2-Gal4 > foi RNAi larvae while increased in Lsp2-Gal4 > foi OE larvae. B Quantitative measurement of (A). C–F Protein levels of both Mmp1 and Mmp2 in the fat body were reduced in Lsp2-Gal4 > foi RNAi larvae while increased in Lsp2-Gal4 > foi OE larvae. Dietary zinc supplementation was able to elevate the expression of Mmps, whereas zinc chelator TPEN can reduce their expression. G, H Both Mmp1 and Mmp2 mRNA levels were significantly down-regulated in Lsp2-Gal4 > foi RNAi larvae, whereas a dramatically up-regulated Mmp2 mRNA level could be detected in Lsp2-Gal4 > foi OE larvae. w1118 flies driven by Lsp2-Gal4 serve as a blank control. All the experiments were conducted independently at least for 3 times. ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed Student’s t test.
Fig. 5
Fig. 5. Retarded fat body cell dissociation caused by Mmps knockdown could be rescued by foi OE.
A, B Fat body cell dissociation was significantly delayed in Lsp2-Gal4 > foi RNAi, whereas a significant advance in fat body cell dissociation was also observed in Lsp2-Gal4 > foi OE. C, D The retarded fat body cell dissociation caused by Mmp1 RNAi could be enhanced by foi RNAi while rescued by foi OE. E, F foi RNAi aggravated the retarded fat body cell dissociation caused by Mmp2 RNAi, while foi OE rescued it. All the experiments were conducted independently at least for 3 times. ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed Student’s t test.
Fig. 6
Fig. 6. Foi knocked down in tumour cells repressed tumour growth and invasion.
A, B Tumour growth and metastasis was observed in RafGOFscrib−/− at 12 days AEL (A), which can be dramatically suppressed by foi RNAi (B). C Foi knockdown inhibited tumour phenotypes of RasV12lgl−/− (n > 200 flies per group). When driven by eye-specific ey-Gal4, the foi RNAi did not cause any noticeable change in the morphology; w1118 flies driven by ey-Gal4 serve as a blank control. D, E Fluorescence micrographs of tumour cells and Mmp1 of eye-antennal disc and VNC were shown. RafGOFscrib−/− flies displayed overgrowth in the cephalic complex (GFP signal), invasive behaviour and massive Mmp1 activation (red signal, white arrowheads). Nuclei were stained with DAPI. F, G Silencing foi in the tumour clones strongly reduced the tumour growth, invasive behaviour and Mmp1 activation.
Fig. 7
Fig. 7. A model to explain the effect of foi on cell dissociation.
In the fat body of Drosophila melanogaster, foi transports zinc into cytosol. In addition to direct activating Mmp activity, the increased zinc level in the cytosol also results in increased zinc in the nucleus which could induce the transcription of Mmp1 and Mmp2. Mmps are involved in the progression of cell dissociation by promoting the degradation and hydrolysis of extracellular matrix components.
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