Limits...
Alternative splicing of human NT5E in cirrhosis and hepatocellular carcinoma produces a negative regulator of ecto-5'-nucleotidase (CD73).

Snider NT, Altshuler PJ, Wan S, Welling TH, Cavalcoli J, Omary MB - Mol. Biol. Cell (2014)

Bottom Line: The two human isoforms exhibited functional differences, such that ectopic expression of canonical CD73 (CD73L) in human HepG2 cells was associated with decreased expression of the proliferation marker Ki67, whereas CD73S expression did not have an effect on Ki67 expression.Furthermore, CD73S complexed with CD73L and promoted proteasome-dependent CD73L degradation.The findings reveal species-specific CD73 regulation, with potential significance to cancer, fibrosis, and other diseases characterized by changes in CD73 expression and function.

View Article: PubMed Central - PubMed

Affiliation: Departments of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48109.

Show MeSH

Related in: MedlinePlus

NT5E-2 is up-regulated in cirrhosis, and HCC and encodes a shorter CD73 protein (CD73S), which is functionally distinct from canonical CD73 (CD73L). (A–C) Relative expression of NT5E-1 and NT5E-2 mRNA in HCC cell lines (A), tumors and adjacent nontumor tissue from HCC surgical specimen obtained from six patients (B), and biopsies from patients with HCV-associated cirrhosis of the liver (C; clinical information on the human HCC and cirrhosis samples provided in Supplemental Table S2). CXCL10 is included as a positive control for HCV cirrhosis samples (Brownell and Polyak, 2013). (D) Protein sequence alignment of the C-termini of CD73L (NP_002517) and CD73S (NP_001191742). The 50 residues (404–453) missing in CD73S form three β-strands and two α-helices and include a catalytic residue (Phe-417). (E) Sequence of the synthetic peptide (ERNNGIHV) used to generate rabbit anti-CD73S antibodies. (F) Detection of total Flag-CD73S and Flag-CD73L protein (bottom) and validation of the CD73S antibody reactivity in Flag immunoprecipitates of transfected HEK293T cell lysates. CD73S and CD73L have predicted molecular weight of 58 and 63 kDa, respectively, but migrate at ∼67 and ∼72 kDa because of glycosylation and the Flag tag. (G) CD73S immunoblot of total tissue lysates from two normal human livers (1, 2) and six HCC paired tumors (T) and adjacent uninvolved liver (L) tissues (HCC 1–6; same as those used in B).
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4263446&req=5

Figure 3: NT5E-2 is up-regulated in cirrhosis, and HCC and encodes a shorter CD73 protein (CD73S), which is functionally distinct from canonical CD73 (CD73L). (A–C) Relative expression of NT5E-1 and NT5E-2 mRNA in HCC cell lines (A), tumors and adjacent nontumor tissue from HCC surgical specimen obtained from six patients (B), and biopsies from patients with HCV-associated cirrhosis of the liver (C; clinical information on the human HCC and cirrhosis samples provided in Supplemental Table S2). CXCL10 is included as a positive control for HCV cirrhosis samples (Brownell and Polyak, 2013). (D) Protein sequence alignment of the C-termini of CD73L (NP_002517) and CD73S (NP_001191742). The 50 residues (404–453) missing in CD73S form three β-strands and two α-helices and include a catalytic residue (Phe-417). (E) Sequence of the synthetic peptide (ERNNGIHV) used to generate rabbit anti-CD73S antibodies. (F) Detection of total Flag-CD73S and Flag-CD73L protein (bottom) and validation of the CD73S antibody reactivity in Flag immunoprecipitates of transfected HEK293T cell lysates. CD73S and CD73L have predicted molecular weight of 58 and 63 kDa, respectively, but migrate at ∼67 and ∼72 kDa because of glycosylation and the Flag tag. (G) CD73S immunoblot of total tissue lysates from two normal human livers (1, 2) and six HCC paired tumors (T) and adjacent uninvolved liver (L) tissues (HCC 1–6; same as those used in B).

Mentions: We found that the two transcripts had comparable relative distribution profiles across 14 normal human tissues (Figure 2A), but NT5E-2 was expressed at significantly lower levels than NT5E-1 (Figure 2B). We also evaluated the relative expression of these two isoforms in nine human cancer cell lines (Figure 2C) and found that the two hepatocellular carcinoma (HCC) cell lines Huh7 and HepG2 expressed among the highest levels of the two transcripts relative to the other tested cell lines (AsPC-1, MCF7, NCI-H118, HT-29, K-562, CCRF-CEM, and MOLT-4).Therefore we focused our subsequent analysis on NT5E expression in normal and diseased livers. Because alternative splicing of genes is known to be altered in disease states (David and Manley, 2010; Singh and Cooper, 2012), we evaluated the expression of NT5E-2 in chronic human liver diseases, including HCV, NAFLD, and HCC. Whereas expression of NT5E-2 did not differ significantly in HCV and NAFLD compared with normal livers, it was dramatically increased in HCC surgical specimens (Figure 2D), pointing to a disease-specific regulation. Furthermore, relative to normal human liver, the expression levels of the NT5E-2 transcript were increased by one to two orders of magnitude in the HCC cell lines (Figure 3A), whereas NT5E-1 expression was either unchanged (Huh7) or decreased (HepG2).


Alternative splicing of human NT5E in cirrhosis and hepatocellular carcinoma produces a negative regulator of ecto-5'-nucleotidase (CD73).

Snider NT, Altshuler PJ, Wan S, Welling TH, Cavalcoli J, Omary MB - Mol. Biol. Cell (2014)

NT5E-2 is up-regulated in cirrhosis, and HCC and encodes a shorter CD73 protein (CD73S), which is functionally distinct from canonical CD73 (CD73L). (A–C) Relative expression of NT5E-1 and NT5E-2 mRNA in HCC cell lines (A), tumors and adjacent nontumor tissue from HCC surgical specimen obtained from six patients (B), and biopsies from patients with HCV-associated cirrhosis of the liver (C; clinical information on the human HCC and cirrhosis samples provided in Supplemental Table S2). CXCL10 is included as a positive control for HCV cirrhosis samples (Brownell and Polyak, 2013). (D) Protein sequence alignment of the C-termini of CD73L (NP_002517) and CD73S (NP_001191742). The 50 residues (404–453) missing in CD73S form three β-strands and two α-helices and include a catalytic residue (Phe-417). (E) Sequence of the synthetic peptide (ERNNGIHV) used to generate rabbit anti-CD73S antibodies. (F) Detection of total Flag-CD73S and Flag-CD73L protein (bottom) and validation of the CD73S antibody reactivity in Flag immunoprecipitates of transfected HEK293T cell lysates. CD73S and CD73L have predicted molecular weight of 58 and 63 kDa, respectively, but migrate at ∼67 and ∼72 kDa because of glycosylation and the Flag tag. (G) CD73S immunoblot of total tissue lysates from two normal human livers (1, 2) and six HCC paired tumors (T) and adjacent uninvolved liver (L) tissues (HCC 1–6; same as those used in B).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4263446&req=5

Figure 3: NT5E-2 is up-regulated in cirrhosis, and HCC and encodes a shorter CD73 protein (CD73S), which is functionally distinct from canonical CD73 (CD73L). (A–C) Relative expression of NT5E-1 and NT5E-2 mRNA in HCC cell lines (A), tumors and adjacent nontumor tissue from HCC surgical specimen obtained from six patients (B), and biopsies from patients with HCV-associated cirrhosis of the liver (C; clinical information on the human HCC and cirrhosis samples provided in Supplemental Table S2). CXCL10 is included as a positive control for HCV cirrhosis samples (Brownell and Polyak, 2013). (D) Protein sequence alignment of the C-termini of CD73L (NP_002517) and CD73S (NP_001191742). The 50 residues (404–453) missing in CD73S form three β-strands and two α-helices and include a catalytic residue (Phe-417). (E) Sequence of the synthetic peptide (ERNNGIHV) used to generate rabbit anti-CD73S antibodies. (F) Detection of total Flag-CD73S and Flag-CD73L protein (bottom) and validation of the CD73S antibody reactivity in Flag immunoprecipitates of transfected HEK293T cell lysates. CD73S and CD73L have predicted molecular weight of 58 and 63 kDa, respectively, but migrate at ∼67 and ∼72 kDa because of glycosylation and the Flag tag. (G) CD73S immunoblot of total tissue lysates from two normal human livers (1, 2) and six HCC paired tumors (T) and adjacent uninvolved liver (L) tissues (HCC 1–6; same as those used in B).
Mentions: We found that the two transcripts had comparable relative distribution profiles across 14 normal human tissues (Figure 2A), but NT5E-2 was expressed at significantly lower levels than NT5E-1 (Figure 2B). We also evaluated the relative expression of these two isoforms in nine human cancer cell lines (Figure 2C) and found that the two hepatocellular carcinoma (HCC) cell lines Huh7 and HepG2 expressed among the highest levels of the two transcripts relative to the other tested cell lines (AsPC-1, MCF7, NCI-H118, HT-29, K-562, CCRF-CEM, and MOLT-4).Therefore we focused our subsequent analysis on NT5E expression in normal and diseased livers. Because alternative splicing of genes is known to be altered in disease states (David and Manley, 2010; Singh and Cooper, 2012), we evaluated the expression of NT5E-2 in chronic human liver diseases, including HCV, NAFLD, and HCC. Whereas expression of NT5E-2 did not differ significantly in HCV and NAFLD compared with normal livers, it was dramatically increased in HCC surgical specimens (Figure 2D), pointing to a disease-specific regulation. Furthermore, relative to normal human liver, the expression levels of the NT5E-2 transcript were increased by one to two orders of magnitude in the HCC cell lines (Figure 3A), whereas NT5E-1 expression was either unchanged (Huh7) or decreased (HepG2).

Bottom Line: The two human isoforms exhibited functional differences, such that ectopic expression of canonical CD73 (CD73L) in human HepG2 cells was associated with decreased expression of the proliferation marker Ki67, whereas CD73S expression did not have an effect on Ki67 expression.Furthermore, CD73S complexed with CD73L and promoted proteasome-dependent CD73L degradation.The findings reveal species-specific CD73 regulation, with potential significance to cancer, fibrosis, and other diseases characterized by changes in CD73 expression and function.

View Article: PubMed Central - PubMed

Affiliation: Departments of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48109.

Show MeSH
Related in: MedlinePlus