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p53- and ERK7-dependent ribosome surveillance response regulates Drosophila insulin-like peptide secretion.

Hasygar K, Hietakangas V - PLoS Genet. (2014)

Bottom Line: A downstream effector of this growth inhibitory response is an atypical MAP kinase ERK7 (ERK8/MAPK15), which is upregulated in the IPCs following impaired ribosome biogenesis as well as starvation.Moreover, we provide evidence that p53 and ERK7 contribute to the inhibition of dILP secretion upon starvation.Thus, we conclude that a cell autonomous ribosome surveillance response, which leads to upregulation of ERK7, inhibits dILP secretion to impede tissue growth under limiting dietary conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences & Institute of Biotechnology, University of Helsinki, Helsinki, Finland.

ABSTRACT
Insulin-like signalling is a conserved mechanism that coordinates animal growth and metabolism with nutrient status. In Drosophila, insulin-producing median neurosecretory cells (IPCs) regulate larval growth by secreting insulin-like peptides (dILPs) in a diet-dependent manner. Previous studies have shown that nutrition affects dILP secretion through humoral signals derived from the fat body. Here we uncover a novel mechanism that operates cell autonomously in the IPCs to regulate dILP secretion. We observed that impairment of ribosome biogenesis specifically in the IPCs strongly inhibits dILP secretion, which consequently leads to reduced body size and a delay in larval development. This response is dependent on p53, a known surveillance factor for ribosome biogenesis. A downstream effector of this growth inhibitory response is an atypical MAP kinase ERK7 (ERK8/MAPK15), which is upregulated in the IPCs following impaired ribosome biogenesis as well as starvation. We show that ERK7 is sufficient and essential to inhibit dILP secretion upon impaired ribosome biogenesis, and it acts epistatically to p53. Moreover, we provide evidence that p53 and ERK7 contribute to the inhibition of dILP secretion upon starvation. Thus, we conclude that a cell autonomous ribosome surveillance response, which leads to upregulation of ERK7, inhibits dILP secretion to impede tissue growth under limiting dietary conditions.

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Disturbed ribosome biogenesis in the IPCs inhibits dILP secretion.(A) Starvation or disturbed ribosome biogenesis by knockdown of Rio1 or Rio2 leads to accumulation of dILP2 in cell bodies of IPCs. (B) Quantification of the immunofluorescence from Fig. 2A. Error bars represent standard deviation (N≥10) (C) Relative mRNA expression of inr in whole larvae is elevated upon starvation or knockdown of Rio1 or Rio2 in the IPCs. Error bars represent standard deviation (N = 3, ≥5 larvae/group). cdk7 was used as an internal reference. (D) IPC-specific knockdown of ribosomal components or genes involved in ribosome biogenesis leads to reduction of body weight. Error bars represent standard deviation (N≥3, ≥10 flies/group). (E, F) Knockdown of Rpl35A or Tsr1 inhibits dILP2 secretion from IPCs. Error bars represent standard deviation (N≥10). (G, H) Depletion of dMyc in the IPCs leads to accumulation of dILP2. Error bars represent standard deviation (N≥10). (I) dMyc knockdown in the IPCs leads to reduced body weight. Error bars represent standard deviation (N≥3, ≥10 flies/group). (J, K) Depletion of Rio2 in the IPCs leads to accumulation of dILP5. Error bars represent standard deviation (N≥10). (L) Rio2 knockdown in the IPCs leads to delay in pupation. Error bars represent standard deviation (N = 4, 30 larvae/group). For all confocal images, IPCs are labelled by GFP (green) and dILP2 or dILP5 is shown as red. *p<0.05, **p<0.01, ***p<0.001 (Student's t-test).
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pgen-1004764-g002: Disturbed ribosome biogenesis in the IPCs inhibits dILP secretion.(A) Starvation or disturbed ribosome biogenesis by knockdown of Rio1 or Rio2 leads to accumulation of dILP2 in cell bodies of IPCs. (B) Quantification of the immunofluorescence from Fig. 2A. Error bars represent standard deviation (N≥10) (C) Relative mRNA expression of inr in whole larvae is elevated upon starvation or knockdown of Rio1 or Rio2 in the IPCs. Error bars represent standard deviation (N = 3, ≥5 larvae/group). cdk7 was used as an internal reference. (D) IPC-specific knockdown of ribosomal components or genes involved in ribosome biogenesis leads to reduction of body weight. Error bars represent standard deviation (N≥3, ≥10 flies/group). (E, F) Knockdown of Rpl35A or Tsr1 inhibits dILP2 secretion from IPCs. Error bars represent standard deviation (N≥10). (G, H) Depletion of dMyc in the IPCs leads to accumulation of dILP2. Error bars represent standard deviation (N≥10). (I) dMyc knockdown in the IPCs leads to reduced body weight. Error bars represent standard deviation (N≥3, ≥10 flies/group). (J, K) Depletion of Rio2 in the IPCs leads to accumulation of dILP5. Error bars represent standard deviation (N≥10). (L) Rio2 knockdown in the IPCs leads to delay in pupation. Error bars represent standard deviation (N = 4, 30 larvae/group). For all confocal images, IPCs are labelled by GFP (green) and dILP2 or dILP5 is shown as red. *p<0.05, **p<0.01, ***p<0.001 (Student's t-test).

Mentions: Secretion of dILPs is a key regulatory level in determining the activity of systemic insulin signalling [5]. dILP2 contributes to the total body weight [26] and its secretion can be assessed by monitoring its accumulation into the cell bodies of IPCs. Accumulation is observed when dILP2 secretion is inhibited upon starvation [5]; (Figure 2A, B). Of the hits, 5 kinases caused significant dILP2 accumulation upon knockdown. These include Cdk12, Adck (CG3608), Pkc98E (Figure S1), as well as two Rio kinases, Rio1 and Rio2 (Figure 2A, B). Rio1 and Rio2 belong to a group of atypical kinases and they have a conserved role in ribosome maturation [27]–[30]. As ribosome biogenesis is a process tightly coupled to nutrient sensing [1], [12], we chose to further explore the role of Rio kinases in dILP secretion. We confirmed that, similarly to starvation, IPC-specific depletion of Rio kinases led to elevated insulin-like receptor (inr) gene expression in the larva (Figure 2C), which is an established readout for reduced peripheral insulin signalling [31]. If the inhibition of dILP2 secretion was due to impaired ribosome biogenesis, we predicted that depletion of other ribosomal genes would phenocopy this effect. Indeed, IPC-specific knockdown of a number of other ribosomal components or genes involved in various steps of ribosome biogenesis led to significantly reduced body size (Figure 2D). To further confirm that the dILP2 secretion phenotypes of Rio kinases were due to impaired ribosome biogenesis, we depleted ribosomal protein Rpl35A and ribosome assembly factor Tsr1 (CG7338) in the IPCs and observed a similar inhibition of dILP2 secretion in both cases (Figure 2E, F). The dILP2-Gal4 driver we used [4] displays activity in the salivary glands, in addition to IPCs. Depletion of Rio2 by a salivary gland specific Sgs3-Gal4 driver [32] caused no growth reduction, ruling out the possibility of nonspecific effects through the salivary glands (Figure S2).


p53- and ERK7-dependent ribosome surveillance response regulates Drosophila insulin-like peptide secretion.

Hasygar K, Hietakangas V - PLoS Genet. (2014)

Disturbed ribosome biogenesis in the IPCs inhibits dILP secretion.(A) Starvation or disturbed ribosome biogenesis by knockdown of Rio1 or Rio2 leads to accumulation of dILP2 in cell bodies of IPCs. (B) Quantification of the immunofluorescence from Fig. 2A. Error bars represent standard deviation (N≥10) (C) Relative mRNA expression of inr in whole larvae is elevated upon starvation or knockdown of Rio1 or Rio2 in the IPCs. Error bars represent standard deviation (N = 3, ≥5 larvae/group). cdk7 was used as an internal reference. (D) IPC-specific knockdown of ribosomal components or genes involved in ribosome biogenesis leads to reduction of body weight. Error bars represent standard deviation (N≥3, ≥10 flies/group). (E, F) Knockdown of Rpl35A or Tsr1 inhibits dILP2 secretion from IPCs. Error bars represent standard deviation (N≥10). (G, H) Depletion of dMyc in the IPCs leads to accumulation of dILP2. Error bars represent standard deviation (N≥10). (I) dMyc knockdown in the IPCs leads to reduced body weight. Error bars represent standard deviation (N≥3, ≥10 flies/group). (J, K) Depletion of Rio2 in the IPCs leads to accumulation of dILP5. Error bars represent standard deviation (N≥10). (L) Rio2 knockdown in the IPCs leads to delay in pupation. Error bars represent standard deviation (N = 4, 30 larvae/group). For all confocal images, IPCs are labelled by GFP (green) and dILP2 or dILP5 is shown as red. *p<0.05, **p<0.01, ***p<0.001 (Student's t-test).
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Show All Figures
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pgen-1004764-g002: Disturbed ribosome biogenesis in the IPCs inhibits dILP secretion.(A) Starvation or disturbed ribosome biogenesis by knockdown of Rio1 or Rio2 leads to accumulation of dILP2 in cell bodies of IPCs. (B) Quantification of the immunofluorescence from Fig. 2A. Error bars represent standard deviation (N≥10) (C) Relative mRNA expression of inr in whole larvae is elevated upon starvation or knockdown of Rio1 or Rio2 in the IPCs. Error bars represent standard deviation (N = 3, ≥5 larvae/group). cdk7 was used as an internal reference. (D) IPC-specific knockdown of ribosomal components or genes involved in ribosome biogenesis leads to reduction of body weight. Error bars represent standard deviation (N≥3, ≥10 flies/group). (E, F) Knockdown of Rpl35A or Tsr1 inhibits dILP2 secretion from IPCs. Error bars represent standard deviation (N≥10). (G, H) Depletion of dMyc in the IPCs leads to accumulation of dILP2. Error bars represent standard deviation (N≥10). (I) dMyc knockdown in the IPCs leads to reduced body weight. Error bars represent standard deviation (N≥3, ≥10 flies/group). (J, K) Depletion of Rio2 in the IPCs leads to accumulation of dILP5. Error bars represent standard deviation (N≥10). (L) Rio2 knockdown in the IPCs leads to delay in pupation. Error bars represent standard deviation (N = 4, 30 larvae/group). For all confocal images, IPCs are labelled by GFP (green) and dILP2 or dILP5 is shown as red. *p<0.05, **p<0.01, ***p<0.001 (Student's t-test).
Mentions: Secretion of dILPs is a key regulatory level in determining the activity of systemic insulin signalling [5]. dILP2 contributes to the total body weight [26] and its secretion can be assessed by monitoring its accumulation into the cell bodies of IPCs. Accumulation is observed when dILP2 secretion is inhibited upon starvation [5]; (Figure 2A, B). Of the hits, 5 kinases caused significant dILP2 accumulation upon knockdown. These include Cdk12, Adck (CG3608), Pkc98E (Figure S1), as well as two Rio kinases, Rio1 and Rio2 (Figure 2A, B). Rio1 and Rio2 belong to a group of atypical kinases and they have a conserved role in ribosome maturation [27]–[30]. As ribosome biogenesis is a process tightly coupled to nutrient sensing [1], [12], we chose to further explore the role of Rio kinases in dILP secretion. We confirmed that, similarly to starvation, IPC-specific depletion of Rio kinases led to elevated insulin-like receptor (inr) gene expression in the larva (Figure 2C), which is an established readout for reduced peripheral insulin signalling [31]. If the inhibition of dILP2 secretion was due to impaired ribosome biogenesis, we predicted that depletion of other ribosomal genes would phenocopy this effect. Indeed, IPC-specific knockdown of a number of other ribosomal components or genes involved in various steps of ribosome biogenesis led to significantly reduced body size (Figure 2D). To further confirm that the dILP2 secretion phenotypes of Rio kinases were due to impaired ribosome biogenesis, we depleted ribosomal protein Rpl35A and ribosome assembly factor Tsr1 (CG7338) in the IPCs and observed a similar inhibition of dILP2 secretion in both cases (Figure 2E, F). The dILP2-Gal4 driver we used [4] displays activity in the salivary glands, in addition to IPCs. Depletion of Rio2 by a salivary gland specific Sgs3-Gal4 driver [32] caused no growth reduction, ruling out the possibility of nonspecific effects through the salivary glands (Figure S2).

Bottom Line: A downstream effector of this growth inhibitory response is an atypical MAP kinase ERK7 (ERK8/MAPK15), which is upregulated in the IPCs following impaired ribosome biogenesis as well as starvation.Moreover, we provide evidence that p53 and ERK7 contribute to the inhibition of dILP secretion upon starvation.Thus, we conclude that a cell autonomous ribosome surveillance response, which leads to upregulation of ERK7, inhibits dILP secretion to impede tissue growth under limiting dietary conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences & Institute of Biotechnology, University of Helsinki, Helsinki, Finland.

ABSTRACT
Insulin-like signalling is a conserved mechanism that coordinates animal growth and metabolism with nutrient status. In Drosophila, insulin-producing median neurosecretory cells (IPCs) regulate larval growth by secreting insulin-like peptides (dILPs) in a diet-dependent manner. Previous studies have shown that nutrition affects dILP secretion through humoral signals derived from the fat body. Here we uncover a novel mechanism that operates cell autonomously in the IPCs to regulate dILP secretion. We observed that impairment of ribosome biogenesis specifically in the IPCs strongly inhibits dILP secretion, which consequently leads to reduced body size and a delay in larval development. This response is dependent on p53, a known surveillance factor for ribosome biogenesis. A downstream effector of this growth inhibitory response is an atypical MAP kinase ERK7 (ERK8/MAPK15), which is upregulated in the IPCs following impaired ribosome biogenesis as well as starvation. We show that ERK7 is sufficient and essential to inhibit dILP secretion upon impaired ribosome biogenesis, and it acts epistatically to p53. Moreover, we provide evidence that p53 and ERK7 contribute to the inhibition of dILP secretion upon starvation. Thus, we conclude that a cell autonomous ribosome surveillance response, which leads to upregulation of ERK7, inhibits dILP secretion to impede tissue growth under limiting dietary conditions.

Show MeSH
Related in: MedlinePlus