Limits...
Nuclear translocation of an ICA512 cytosolic fragment couples granule exocytosis and insulin expression in {beta}-cells.

Trajkovski M, Mziaut H, Altkrüger A, Ouwendijk J, Knoch KP, Müller S, Solimena M - J. Cell Biol. (2004)

Bottom Line: Islet cell autoantigen 512 (ICA512)/IA-2 is a receptor tyrosine phosphatase-like protein associated with the insulin secretory granules (SGs) of pancreatic beta-cells.This cleavage occurs at the plasma membrane and generates an ICA512 cytosolic fragment that is targeted to the nucleus, where it binds the E3-SUMO ligase protein inhibitor of activated signal transducer and activator of transcription-y (PIASy) and up-regulates insulin expression.Accordingly, this novel pathway directly links regulated exocytosis of SGs and control of gene expression in beta-cells, whose impaired insulin production and secretion causes diabetes.

View Article: PubMed Central - PubMed

Affiliation: Experimental Diabetology, Carl Gustav Carus Medical School, Dresden University of Technology, Dresden, Germany.

ABSTRACT
Islet cell autoantigen 512 (ICA512)/IA-2 is a receptor tyrosine phosphatase-like protein associated with the insulin secretory granules (SGs) of pancreatic beta-cells. Here, we show that exocytosis of SGs and insertion of ICA512 in the plasma membrane promotes the Ca(2+)-dependent cleavage of ICA512 cytoplasmic domain by mu-calpain. This cleavage occurs at the plasma membrane and generates an ICA512 cytosolic fragment that is targeted to the nucleus, where it binds the E3-SUMO ligase protein inhibitor of activated signal transducer and activator of transcription-y (PIASy) and up-regulates insulin expression. Accordingly, this novel pathway directly links regulated exocytosis of SGs and control of gene expression in beta-cells, whose impaired insulin production and secretion causes diabetes.

Show MeSH

Related in: MedlinePlus

ICA512-CCF promotes insulin gene expression. (A) Confocal microscopy on INS-1 cells transiently transfected with ICA512 (659–979)-GFP and counterstained with DAPI. Bar, 10 μm. (B) Western blot with goat anti-GFP (top) and anti–γ-tubulin (bottom) antibodies on 6 μg of nuclear protein from INS-1 cells transiently transfected with ICA512(659–979)-GFP and incubated with resting or stimulating buffer for 105 min after 1 h at rest. (C) Insulin mRNA levels quantified by real-time PCR in INS-1 cells that were either electroporated only or transfected with 4 μg of vector encoding GFP, ICA512-GFP, or ICA512(659–979)-GFP and kept at rest for 1 h. The results are from three independent experiments, each in triplicate. (D) Insulin mRNA levels quantified by real-time PCR in INS-1 cells which were either electroporated only or transfected with 0.5–4 μg ICA512(659–979)-GFP and collected after 4 d in culture with 11 mM glucose. The results are from four independent experiments, each in triplicate. In C and D, the levels of insulin mRNA in electroporated cells were equaled to 100%. In D, ICA512(659–979)-GFP and γ-tubulin levels were assessed by Western blotting with goat anti-GFP (middle) and anti-γ-tubulin (bottom) antibodies. (E) Insulin mRNA levels quantified by real-time PCR in INS-1 cells transfected either with scrambled or anti–μ-calpain siRNA oligos 1 + 2. The results are from three independent experiments, each in triplicate. The level of insulin mRNA in cells transfected with the scrambled oligos was equaled to 100%. Error bars (C, D, and E) show mean + SD. (F) Insulin mRNA levels quantified by real-time PCR in INS-1 cells that were at rest (2.8 mM glucose and 5 mM KCl) for 1 h, then in resting or stimulating buffer, with or without 40 μM calpeptin, for up to 6 h (pulse), and finally in resting buffer for up to 24 h (chase). Total RNA was collected at 0-, 2-, and 6-h time points of the pulse and then at 2-, 6-, 12-, and 24-h time points of the chase. The results are from three independent experiments, each in triplicate. The level of insulin mRNA in resting INS-1 cells was equaled to 100%. Error bars show mean ± SD.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172607&req=5

fig7: ICA512-CCF promotes insulin gene expression. (A) Confocal microscopy on INS-1 cells transiently transfected with ICA512 (659–979)-GFP and counterstained with DAPI. Bar, 10 μm. (B) Western blot with goat anti-GFP (top) and anti–γ-tubulin (bottom) antibodies on 6 μg of nuclear protein from INS-1 cells transiently transfected with ICA512(659–979)-GFP and incubated with resting or stimulating buffer for 105 min after 1 h at rest. (C) Insulin mRNA levels quantified by real-time PCR in INS-1 cells that were either electroporated only or transfected with 4 μg of vector encoding GFP, ICA512-GFP, or ICA512(659–979)-GFP and kept at rest for 1 h. The results are from three independent experiments, each in triplicate. (D) Insulin mRNA levels quantified by real-time PCR in INS-1 cells which were either electroporated only or transfected with 0.5–4 μg ICA512(659–979)-GFP and collected after 4 d in culture with 11 mM glucose. The results are from four independent experiments, each in triplicate. In C and D, the levels of insulin mRNA in electroporated cells were equaled to 100%. In D, ICA512(659–979)-GFP and γ-tubulin levels were assessed by Western blotting with goat anti-GFP (middle) and anti-γ-tubulin (bottom) antibodies. (E) Insulin mRNA levels quantified by real-time PCR in INS-1 cells transfected either with scrambled or anti–μ-calpain siRNA oligos 1 + 2. The results are from three independent experiments, each in triplicate. The level of insulin mRNA in cells transfected with the scrambled oligos was equaled to 100%. Error bars (C, D, and E) show mean + SD. (F) Insulin mRNA levels quantified by real-time PCR in INS-1 cells that were at rest (2.8 mM glucose and 5 mM KCl) for 1 h, then in resting or stimulating buffer, with or without 40 μM calpeptin, for up to 6 h (pulse), and finally in resting buffer for up to 24 h (chase). Total RNA was collected at 0-, 2-, and 6-h time points of the pulse and then at 2-, 6-, 12-, and 24-h time points of the chase. The results are from three independent experiments, each in triplicate. The level of insulin mRNA in resting INS-1 cells was equaled to 100%. Error bars show mean ± SD.

Mentions: To test whether or not ICA512-CCF is a retrograde signal that couples regulated secretion and gene expression, possibly through PIASy, INS-1 cells were transiently transfected with the ICA512 cytoplasmic fragment generated in vitro by μ-calpain (Ort et al., 2001; Fig. S1), which was tagged at its COOH terminus with GFP (ICA512(659–979)-GFP). Notably, ICA512(659–979) includes the catalytically inactive PTP domain. Confocal microscopy (Fig. 7 A) and Western blotting (Fig. 7 B) show that the nuclei of resting and stimulated cells contain comparable amounts of ICA512(659–979). Strikingly, insulin mRNA levels are increased by 32 and 93% in resting INS-1 cells expressing ICA512-GFP or ICA512(659–979)-GFP, respectively, but are not changed by the expression of GFP alone (Fig. 7 C). The higher levels of insulin mRNA in ICA512-GFP cells can be attributed to an increased generation of ICA512-CCF-GFP, as insulin mRNA is enhanced in parallel with the expression of ICA512(659–979)-GFP (Fig. 7 D). Accordingly, knockdown of μ-calpain decreases insulin mRNA (Fig. 7 E). Likewise, calpeptin has an adverse effect on insulin mRNA in stimulated cells (Fig. 7 F). In these experiments, cells were either kept at rest or stimulated for up to 6 h, with or without 40 μM calpeptin, and then returned to resting medium for up to 24 h. Maximal levels of insulin mRNAs are present 2 h after the 6-h stimulation period. However, in calpeptin-treated cells, insulin mRNA is reduced by ∼40% at this time point as well as at any later time point compared with cells stimulated in the absence of calpeptin. The magnitude of this decrease correlates well with the degree of calpeptin-mediated inhibition of ICA512-TMF cleavage (Fig. 1 B).


Nuclear translocation of an ICA512 cytosolic fragment couples granule exocytosis and insulin expression in {beta}-cells.

Trajkovski M, Mziaut H, Altkrüger A, Ouwendijk J, Knoch KP, Müller S, Solimena M - J. Cell Biol. (2004)

ICA512-CCF promotes insulin gene expression. (A) Confocal microscopy on INS-1 cells transiently transfected with ICA512 (659–979)-GFP and counterstained with DAPI. Bar, 10 μm. (B) Western blot with goat anti-GFP (top) and anti–γ-tubulin (bottom) antibodies on 6 μg of nuclear protein from INS-1 cells transiently transfected with ICA512(659–979)-GFP and incubated with resting or stimulating buffer for 105 min after 1 h at rest. (C) Insulin mRNA levels quantified by real-time PCR in INS-1 cells that were either electroporated only or transfected with 4 μg of vector encoding GFP, ICA512-GFP, or ICA512(659–979)-GFP and kept at rest for 1 h. The results are from three independent experiments, each in triplicate. (D) Insulin mRNA levels quantified by real-time PCR in INS-1 cells which were either electroporated only or transfected with 0.5–4 μg ICA512(659–979)-GFP and collected after 4 d in culture with 11 mM glucose. The results are from four independent experiments, each in triplicate. In C and D, the levels of insulin mRNA in electroporated cells were equaled to 100%. In D, ICA512(659–979)-GFP and γ-tubulin levels were assessed by Western blotting with goat anti-GFP (middle) and anti-γ-tubulin (bottom) antibodies. (E) Insulin mRNA levels quantified by real-time PCR in INS-1 cells transfected either with scrambled or anti–μ-calpain siRNA oligos 1 + 2. The results are from three independent experiments, each in triplicate. The level of insulin mRNA in cells transfected with the scrambled oligos was equaled to 100%. Error bars (C, D, and E) show mean + SD. (F) Insulin mRNA levels quantified by real-time PCR in INS-1 cells that were at rest (2.8 mM glucose and 5 mM KCl) for 1 h, then in resting or stimulating buffer, with or without 40 μM calpeptin, for up to 6 h (pulse), and finally in resting buffer for up to 24 h (chase). Total RNA was collected at 0-, 2-, and 6-h time points of the pulse and then at 2-, 6-, 12-, and 24-h time points of the chase. The results are from three independent experiments, each in triplicate. The level of insulin mRNA in resting INS-1 cells was equaled to 100%. Error bars show mean ± SD.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172607&req=5

fig7: ICA512-CCF promotes insulin gene expression. (A) Confocal microscopy on INS-1 cells transiently transfected with ICA512 (659–979)-GFP and counterstained with DAPI. Bar, 10 μm. (B) Western blot with goat anti-GFP (top) and anti–γ-tubulin (bottom) antibodies on 6 μg of nuclear protein from INS-1 cells transiently transfected with ICA512(659–979)-GFP and incubated with resting or stimulating buffer for 105 min after 1 h at rest. (C) Insulin mRNA levels quantified by real-time PCR in INS-1 cells that were either electroporated only or transfected with 4 μg of vector encoding GFP, ICA512-GFP, or ICA512(659–979)-GFP and kept at rest for 1 h. The results are from three independent experiments, each in triplicate. (D) Insulin mRNA levels quantified by real-time PCR in INS-1 cells which were either electroporated only or transfected with 0.5–4 μg ICA512(659–979)-GFP and collected after 4 d in culture with 11 mM glucose. The results are from four independent experiments, each in triplicate. In C and D, the levels of insulin mRNA in electroporated cells were equaled to 100%. In D, ICA512(659–979)-GFP and γ-tubulin levels were assessed by Western blotting with goat anti-GFP (middle) and anti-γ-tubulin (bottom) antibodies. (E) Insulin mRNA levels quantified by real-time PCR in INS-1 cells transfected either with scrambled or anti–μ-calpain siRNA oligos 1 + 2. The results are from three independent experiments, each in triplicate. The level of insulin mRNA in cells transfected with the scrambled oligos was equaled to 100%. Error bars (C, D, and E) show mean + SD. (F) Insulin mRNA levels quantified by real-time PCR in INS-1 cells that were at rest (2.8 mM glucose and 5 mM KCl) for 1 h, then in resting or stimulating buffer, with or without 40 μM calpeptin, for up to 6 h (pulse), and finally in resting buffer for up to 24 h (chase). Total RNA was collected at 0-, 2-, and 6-h time points of the pulse and then at 2-, 6-, 12-, and 24-h time points of the chase. The results are from three independent experiments, each in triplicate. The level of insulin mRNA in resting INS-1 cells was equaled to 100%. Error bars show mean ± SD.
Mentions: To test whether or not ICA512-CCF is a retrograde signal that couples regulated secretion and gene expression, possibly through PIASy, INS-1 cells were transiently transfected with the ICA512 cytoplasmic fragment generated in vitro by μ-calpain (Ort et al., 2001; Fig. S1), which was tagged at its COOH terminus with GFP (ICA512(659–979)-GFP). Notably, ICA512(659–979) includes the catalytically inactive PTP domain. Confocal microscopy (Fig. 7 A) and Western blotting (Fig. 7 B) show that the nuclei of resting and stimulated cells contain comparable amounts of ICA512(659–979). Strikingly, insulin mRNA levels are increased by 32 and 93% in resting INS-1 cells expressing ICA512-GFP or ICA512(659–979)-GFP, respectively, but are not changed by the expression of GFP alone (Fig. 7 C). The higher levels of insulin mRNA in ICA512-GFP cells can be attributed to an increased generation of ICA512-CCF-GFP, as insulin mRNA is enhanced in parallel with the expression of ICA512(659–979)-GFP (Fig. 7 D). Accordingly, knockdown of μ-calpain decreases insulin mRNA (Fig. 7 E). Likewise, calpeptin has an adverse effect on insulin mRNA in stimulated cells (Fig. 7 F). In these experiments, cells were either kept at rest or stimulated for up to 6 h, with or without 40 μM calpeptin, and then returned to resting medium for up to 24 h. Maximal levels of insulin mRNAs are present 2 h after the 6-h stimulation period. However, in calpeptin-treated cells, insulin mRNA is reduced by ∼40% at this time point as well as at any later time point compared with cells stimulated in the absence of calpeptin. The magnitude of this decrease correlates well with the degree of calpeptin-mediated inhibition of ICA512-TMF cleavage (Fig. 1 B).

Bottom Line: Islet cell autoantigen 512 (ICA512)/IA-2 is a receptor tyrosine phosphatase-like protein associated with the insulin secretory granules (SGs) of pancreatic beta-cells.This cleavage occurs at the plasma membrane and generates an ICA512 cytosolic fragment that is targeted to the nucleus, where it binds the E3-SUMO ligase protein inhibitor of activated signal transducer and activator of transcription-y (PIASy) and up-regulates insulin expression.Accordingly, this novel pathway directly links regulated exocytosis of SGs and control of gene expression in beta-cells, whose impaired insulin production and secretion causes diabetes.

View Article: PubMed Central - PubMed

Affiliation: Experimental Diabetology, Carl Gustav Carus Medical School, Dresden University of Technology, Dresden, Germany.

ABSTRACT
Islet cell autoantigen 512 (ICA512)/IA-2 is a receptor tyrosine phosphatase-like protein associated with the insulin secretory granules (SGs) of pancreatic beta-cells. Here, we show that exocytosis of SGs and insertion of ICA512 in the plasma membrane promotes the Ca(2+)-dependent cleavage of ICA512 cytoplasmic domain by mu-calpain. This cleavage occurs at the plasma membrane and generates an ICA512 cytosolic fragment that is targeted to the nucleus, where it binds the E3-SUMO ligase protein inhibitor of activated signal transducer and activator of transcription-y (PIASy) and up-regulates insulin expression. Accordingly, this novel pathway directly links regulated exocytosis of SGs and control of gene expression in beta-cells, whose impaired insulin production and secretion causes diabetes.

Show MeSH
Related in: MedlinePlus