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Notch1 activation up-regulates pancreatic and duodenal homeobox-1.

Liu SH, Zhou G, Yu J, Wu J, Nemunaitis J, Senzer N, Dawson D, Li M, Fisher WE, Brunicardi FC - Genes (Basel) (2013)

Bottom Line: We report here that overexpression of Notch1 intracellular domain (NICD), an activated form of Notch1, enhanced PDX-1 expression in both PDX-1 stable HEK293 cells and mouse insulinoma β-TC-6 cells, while NICD shRNA inhibited the enhancing effect.NICD-enhanced PDX-1 expression was accompanied by increased insulin expression/secretion and cell proliferation in β-TC-6 cells, which was reversed by NICD shRNA.Moreover, expression levels of NICD were correlated with those of PDX-1 in human pancreatic neuroendocrine tumor.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, David Geffen School of Medicine at University of California, Los Angeles, CA 90095, USA. sliu@mednet.ucla.edu.

ABSTRACT
Transcription factor pancreatic and duodenal homeobox-1 (PDX-1) plays an essential role in pancreatic development, β-cell differentiation, maintenance of normal β-cell function and tumorigenesis. PDX-1 expression is tightly controlled through a variety of mechanisms under different cellular contexts. We report here that overexpression of Notch1 intracellular domain (NICD), an activated form of Notch1, enhanced PDX-1 expression in both PDX-1 stable HEK293 cells and mouse insulinoma β-TC-6 cells, while NICD shRNA inhibited the enhancing effect. NICD-enhanced PDX-1 expression was accompanied by increased insulin expression/secretion and cell proliferation in β-TC-6 cells, which was reversed by NICD shRNA. Cre activation-induced specific expression of NICD in islet β cells of transgenic βNICD+/+ mice induced increased expression of PDX-1, insulin and proliferating cell nuclear antigen (PCNA) and decreased expression of p27 with accompanied fasting hyperinsulinemia and hypoglycemia and altered responses to intraperitoneal glucose tolerance test. Systemically delivered NICD shRNA suppressed islet expression of PDX-1 and reversed the hypoglycemia and hyperinsulinemia. Moreover, expression levels of NICD were correlated with those of PDX-1 in human pancreatic neuroendocrine tumor. Thus, Notch1 acts as a positive regulator for PDX-1 expression, cooperates with PDX-1 in the development of insulin overexpression and islet cell neoplasia and represents a potential therapeutic target for islet neoplasia.

No MeSH data available.


Related in: MedlinePlus

NICD-enhanced PDX-1 expression is accompanied by increased insulin expression/secretion and cell proliferation in β-TC-6 cells. mNICD or shRNAmNICD was transfected into β-TC-6 cells using Lipofectamine 2000. (A) Immunohistochemistry analyses were performed using antibodies against NICD, PDX-1 (1:200) or insulin (1:75). Fluorescence was developed using cy3- or FITC-conjugated secondary antibody. Photomicrographs were taken under 100× magnification. (B) Immunostaining for the expression of NICD, PDX-1 and insulin was semi-quantified using ImageJ (∗ indicates p < 0.05 showing significant difference). (C) Forty-eight (48) hours after transfection, β-TC-6 cells were washed twice with Krebs-Ringer bicarbonate (KRB) buffer and incubated in KRB-BSA for 1 hour. The cells were then added a variety of concentrations of glucose as indicated for 4 hours. After incubation, the media were collected and centrifuged at 600 g for 5 minutes. The insulin concentrations in the media were measured by ELISA assay (∗ indicates p < 0.05: Mock vs. mNICD transfection; p < 0.05: Mock vs. shRNAmNICD transfection, showing significant difference). (D) Twenty four (24) hours after transfection, β-TC-6 cells were replated into 96-well cell culture plates at 5 × 103 cells/well. Cell proliferation was determined by MTS assay (Promega, Madison, WI, UAS) at 24, 48, and 72 hour after transfection. The absorbance was read using a Multiskan EX plate reader (Thermo Electronic Corp, Franklin, MA, USA) at 492 nm.
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genes-04-00358-f002: NICD-enhanced PDX-1 expression is accompanied by increased insulin expression/secretion and cell proliferation in β-TC-6 cells. mNICD or shRNAmNICD was transfected into β-TC-6 cells using Lipofectamine 2000. (A) Immunohistochemistry analyses were performed using antibodies against NICD, PDX-1 (1:200) or insulin (1:75). Fluorescence was developed using cy3- or FITC-conjugated secondary antibody. Photomicrographs were taken under 100× magnification. (B) Immunostaining for the expression of NICD, PDX-1 and insulin was semi-quantified using ImageJ (∗ indicates p < 0.05 showing significant difference). (C) Forty-eight (48) hours after transfection, β-TC-6 cells were washed twice with Krebs-Ringer bicarbonate (KRB) buffer and incubated in KRB-BSA for 1 hour. The cells were then added a variety of concentrations of glucose as indicated for 4 hours. After incubation, the media were collected and centrifuged at 600 g for 5 minutes. The insulin concentrations in the media were measured by ELISA assay (∗ indicates p < 0.05: Mock vs. mNICD transfection; p < 0.05: Mock vs. shRNAmNICD transfection, showing significant difference). (D) Twenty four (24) hours after transfection, β-TC-6 cells were replated into 96-well cell culture plates at 5 × 103 cells/well. Cell proliferation was determined by MTS assay (Promega, Madison, WI, UAS) at 24, 48, and 72 hour after transfection. The absorbance was read using a Multiskan EX plate reader (Thermo Electronic Corp, Franklin, MA, USA) at 492 nm.

Mentions: PDX-1 plays an essential role in insulin expression and secretion [3,4] and cell proliferation [54]. To determine the functional relevance of the positive regulation of PDX-1 by NICD, we sought to examine the effect of NICD on PDX-1-mediated insulin expression/secretion and cell proliferation in β-TC-6 cells. β-TC-6 cells were transfected with NICD or shRNANICD. Immunohistochemistry analysis showed that overexpression of NICD further increased NICD expression in β-TC-6 cells, while overexpression of shRNANICD inhibited NICD expression (Figure 2A, top panel). As expected, PDX-1 expression level was increased by transfected NICD and decreased by transfected shRNANICD (Figure 2A, middle panel). Moreover, we found that NICD-enhanced PDX-1 expression was accompanied by increased insulin expression, and NICD knockdown-induced inhibition of PDX-1 expression was accompanied by decreased insulin expression (Figure 2A, bottom panel). Insulin ELISA analysis showed that NICD overexpression significantly increased glucose stimulated insulin secretion (GSIS) in comparison to mock transfection, whereas shRNANICD led to significant inhibition of insulin secretion in β-TC-6 cells (Figure 2B). By performing MTS assays, we found that, at 24, 48 and 72 hour after transfection, overexpression of NICD resulted in significant increases of β-TC-6 cell proliferation by 174 ± 18.2%, 185 ± 30.8% and 166 ± 21.3%, respectively, as compared to the mock transfection (Figure 2C). Conversely, transfection of shRNANICD led to significant decreases of cell proliferation by 63 ± 15.3%, 49 ± 8.8% and 56 ± 12.6% at each time point (Figure 2C). All these data indicate that Notch1 activation exerts a positive effect on insulin expression/secretion and cell proliferation likely through a mechanism of up-regulating PDX-1 expression in β-TC-6 cells.


Notch1 activation up-regulates pancreatic and duodenal homeobox-1.

Liu SH, Zhou G, Yu J, Wu J, Nemunaitis J, Senzer N, Dawson D, Li M, Fisher WE, Brunicardi FC - Genes (Basel) (2013)

NICD-enhanced PDX-1 expression is accompanied by increased insulin expression/secretion and cell proliferation in β-TC-6 cells. mNICD or shRNAmNICD was transfected into β-TC-6 cells using Lipofectamine 2000. (A) Immunohistochemistry analyses were performed using antibodies against NICD, PDX-1 (1:200) or insulin (1:75). Fluorescence was developed using cy3- or FITC-conjugated secondary antibody. Photomicrographs were taken under 100× magnification. (B) Immunostaining for the expression of NICD, PDX-1 and insulin was semi-quantified using ImageJ (∗ indicates p < 0.05 showing significant difference). (C) Forty-eight (48) hours after transfection, β-TC-6 cells were washed twice with Krebs-Ringer bicarbonate (KRB) buffer and incubated in KRB-BSA for 1 hour. The cells were then added a variety of concentrations of glucose as indicated for 4 hours. After incubation, the media were collected and centrifuged at 600 g for 5 minutes. The insulin concentrations in the media were measured by ELISA assay (∗ indicates p < 0.05: Mock vs. mNICD transfection; p < 0.05: Mock vs. shRNAmNICD transfection, showing significant difference). (D) Twenty four (24) hours after transfection, β-TC-6 cells were replated into 96-well cell culture plates at 5 × 103 cells/well. Cell proliferation was determined by MTS assay (Promega, Madison, WI, UAS) at 24, 48, and 72 hour after transfection. The absorbance was read using a Multiskan EX plate reader (Thermo Electronic Corp, Franklin, MA, USA) at 492 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3924823&req=5

genes-04-00358-f002: NICD-enhanced PDX-1 expression is accompanied by increased insulin expression/secretion and cell proliferation in β-TC-6 cells. mNICD or shRNAmNICD was transfected into β-TC-6 cells using Lipofectamine 2000. (A) Immunohistochemistry analyses were performed using antibodies against NICD, PDX-1 (1:200) or insulin (1:75). Fluorescence was developed using cy3- or FITC-conjugated secondary antibody. Photomicrographs were taken under 100× magnification. (B) Immunostaining for the expression of NICD, PDX-1 and insulin was semi-quantified using ImageJ (∗ indicates p < 0.05 showing significant difference). (C) Forty-eight (48) hours after transfection, β-TC-6 cells were washed twice with Krebs-Ringer bicarbonate (KRB) buffer and incubated in KRB-BSA for 1 hour. The cells were then added a variety of concentrations of glucose as indicated for 4 hours. After incubation, the media were collected and centrifuged at 600 g for 5 minutes. The insulin concentrations in the media were measured by ELISA assay (∗ indicates p < 0.05: Mock vs. mNICD transfection; p < 0.05: Mock vs. shRNAmNICD transfection, showing significant difference). (D) Twenty four (24) hours after transfection, β-TC-6 cells were replated into 96-well cell culture plates at 5 × 103 cells/well. Cell proliferation was determined by MTS assay (Promega, Madison, WI, UAS) at 24, 48, and 72 hour after transfection. The absorbance was read using a Multiskan EX plate reader (Thermo Electronic Corp, Franklin, MA, USA) at 492 nm.
Mentions: PDX-1 plays an essential role in insulin expression and secretion [3,4] and cell proliferation [54]. To determine the functional relevance of the positive regulation of PDX-1 by NICD, we sought to examine the effect of NICD on PDX-1-mediated insulin expression/secretion and cell proliferation in β-TC-6 cells. β-TC-6 cells were transfected with NICD or shRNANICD. Immunohistochemistry analysis showed that overexpression of NICD further increased NICD expression in β-TC-6 cells, while overexpression of shRNANICD inhibited NICD expression (Figure 2A, top panel). As expected, PDX-1 expression level was increased by transfected NICD and decreased by transfected shRNANICD (Figure 2A, middle panel). Moreover, we found that NICD-enhanced PDX-1 expression was accompanied by increased insulin expression, and NICD knockdown-induced inhibition of PDX-1 expression was accompanied by decreased insulin expression (Figure 2A, bottom panel). Insulin ELISA analysis showed that NICD overexpression significantly increased glucose stimulated insulin secretion (GSIS) in comparison to mock transfection, whereas shRNANICD led to significant inhibition of insulin secretion in β-TC-6 cells (Figure 2B). By performing MTS assays, we found that, at 24, 48 and 72 hour after transfection, overexpression of NICD resulted in significant increases of β-TC-6 cell proliferation by 174 ± 18.2%, 185 ± 30.8% and 166 ± 21.3%, respectively, as compared to the mock transfection (Figure 2C). Conversely, transfection of shRNANICD led to significant decreases of cell proliferation by 63 ± 15.3%, 49 ± 8.8% and 56 ± 12.6% at each time point (Figure 2C). All these data indicate that Notch1 activation exerts a positive effect on insulin expression/secretion and cell proliferation likely through a mechanism of up-regulating PDX-1 expression in β-TC-6 cells.

Bottom Line: We report here that overexpression of Notch1 intracellular domain (NICD), an activated form of Notch1, enhanced PDX-1 expression in both PDX-1 stable HEK293 cells and mouse insulinoma β-TC-6 cells, while NICD shRNA inhibited the enhancing effect.NICD-enhanced PDX-1 expression was accompanied by increased insulin expression/secretion and cell proliferation in β-TC-6 cells, which was reversed by NICD shRNA.Moreover, expression levels of NICD were correlated with those of PDX-1 in human pancreatic neuroendocrine tumor.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, David Geffen School of Medicine at University of California, Los Angeles, CA 90095, USA. sliu@mednet.ucla.edu.

ABSTRACT
Transcription factor pancreatic and duodenal homeobox-1 (PDX-1) plays an essential role in pancreatic development, β-cell differentiation, maintenance of normal β-cell function and tumorigenesis. PDX-1 expression is tightly controlled through a variety of mechanisms under different cellular contexts. We report here that overexpression of Notch1 intracellular domain (NICD), an activated form of Notch1, enhanced PDX-1 expression in both PDX-1 stable HEK293 cells and mouse insulinoma β-TC-6 cells, while NICD shRNA inhibited the enhancing effect. NICD-enhanced PDX-1 expression was accompanied by increased insulin expression/secretion and cell proliferation in β-TC-6 cells, which was reversed by NICD shRNA. Cre activation-induced specific expression of NICD in islet β cells of transgenic βNICD+/+ mice induced increased expression of PDX-1, insulin and proliferating cell nuclear antigen (PCNA) and decreased expression of p27 with accompanied fasting hyperinsulinemia and hypoglycemia and altered responses to intraperitoneal glucose tolerance test. Systemically delivered NICD shRNA suppressed islet expression of PDX-1 and reversed the hypoglycemia and hyperinsulinemia. Moreover, expression levels of NICD were correlated with those of PDX-1 in human pancreatic neuroendocrine tumor. Thus, Notch1 acts as a positive regulator for PDX-1 expression, cooperates with PDX-1 in the development of insulin overexpression and islet cell neoplasia and represents a potential therapeutic target for islet neoplasia.

No MeSH data available.


Related in: MedlinePlus