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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.

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Molecular and functional characteristics of CD73S. (A) Coimmunofluorescence analysis of the proliferation marker Ki67 (red) and Flag-CD73S/L (green) in transfected HepG2 cells. Blue, 4′,6-diamidino-2-phenylindole (DAPI); bar, 20 μm. (B) Quantification of Ki67 immunofluorescence staining in CD73-expressing cells relative to neighbor untransfected cells (30 CD73+ cells and 150 CD73− untransfected cells were counted per condition). ****p < 0.0001, unpaired t test. (C) Measurement of 5′-nucleotidase activity in transfected HEK293T cell lysates. Samples expressing equal protein levels (bottom) were analyzed in triplicate. (D) Treatment of CD73-transfected HEK293T cell lysates with peptide-N-glycosidase F results in similar deglycosylation of both isoforms. (E) Immunofluorescence-based localization of Flag-CD73L or Flag-CD73S in primary mouse hepatocytes. Blue, DAPI; bar, 20 μm. (F) Flag immunoblot of Flag-CD73L– and -CD73S–expressing HEK293T total cell lysates analyzed under reducing (+β-mercaptoethanol, βME) or nonreducing (–βME) conditions, showing that CD73S does not dimerize. (G) Coomassie-stained gel of Flag immunoprecipitates of the nonreduced samples shown in F. Asterisk denotes a unique protein present in CD73S immunoprecipitates, identified by mass spectrometry as calnexin. (H) Biochemical validation that CD73S coimmunoprecipitates with calnexin.
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Figure 5: Molecular and functional characteristics of CD73S. (A) Coimmunofluorescence analysis of the proliferation marker Ki67 (red) and Flag-CD73S/L (green) in transfected HepG2 cells. Blue, 4′,6-diamidino-2-phenylindole (DAPI); bar, 20 μm. (B) Quantification of Ki67 immunofluorescence staining in CD73-expressing cells relative to neighbor untransfected cells (30 CD73+ cells and 150 CD73− untransfected cells were counted per condition). ****p < 0.0001, unpaired t test. (C) Measurement of 5′-nucleotidase activity in transfected HEK293T cell lysates. Samples expressing equal protein levels (bottom) were analyzed in triplicate. (D) Treatment of CD73-transfected HEK293T cell lysates with peptide-N-glycosidase F results in similar deglycosylation of both isoforms. (E) Immunofluorescence-based localization of Flag-CD73L or Flag-CD73S in primary mouse hepatocytes. Blue, DAPI; bar, 20 μm. (F) Flag immunoblot of Flag-CD73L– and -CD73S–expressing HEK293T total cell lysates analyzed under reducing (+β-mercaptoethanol, βME) or nonreducing (–βME) conditions, showing that CD73S does not dimerize. (G) Coomassie-stained gel of Flag immunoprecipitates of the nonreduced samples shown in F. Asterisk denotes a unique protein present in CD73S immunoprecipitates, identified by mass spectrometry as calnexin. (H) Biochemical validation that CD73S coimmunoprecipitates with calnexin.

Mentions: To determine whether CD73S and CD73L exhibit functional differences, we transfected each isoform into HepG2 cells and monitored the expression of the cell proliferation marker Ki67. The cells expressing CD73L had a significant decrease in Ki67 staining, whereas CD73S expression did not significantly alter Ki67 expression relative to neighboring untransfected cells (Figure 5, A and B). Therefore CD73L and CD73S exhibit functional differences with respect to their ability to modulate the proliferation potential of cultured human HCC cells.


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)

Molecular and functional characteristics of CD73S. (A) Coimmunofluorescence analysis of the proliferation marker Ki67 (red) and Flag-CD73S/L (green) in transfected HepG2 cells. Blue, 4′,6-diamidino-2-phenylindole (DAPI); bar, 20 μm. (B) Quantification of Ki67 immunofluorescence staining in CD73-expressing cells relative to neighbor untransfected cells (30 CD73+ cells and 150 CD73− untransfected cells were counted per condition). ****p < 0.0001, unpaired t test. (C) Measurement of 5′-nucleotidase activity in transfected HEK293T cell lysates. Samples expressing equal protein levels (bottom) were analyzed in triplicate. (D) Treatment of CD73-transfected HEK293T cell lysates with peptide-N-glycosidase F results in similar deglycosylation of both isoforms. (E) Immunofluorescence-based localization of Flag-CD73L or Flag-CD73S in primary mouse hepatocytes. Blue, DAPI; bar, 20 μm. (F) Flag immunoblot of Flag-CD73L– and -CD73S–expressing HEK293T total cell lysates analyzed under reducing (+β-mercaptoethanol, βME) or nonreducing (–βME) conditions, showing that CD73S does not dimerize. (G) Coomassie-stained gel of Flag immunoprecipitates of the nonreduced samples shown in F. Asterisk denotes a unique protein present in CD73S immunoprecipitates, identified by mass spectrometry as calnexin. (H) Biochemical validation that CD73S coimmunoprecipitates with calnexin.
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Figure 5: Molecular and functional characteristics of CD73S. (A) Coimmunofluorescence analysis of the proliferation marker Ki67 (red) and Flag-CD73S/L (green) in transfected HepG2 cells. Blue, 4′,6-diamidino-2-phenylindole (DAPI); bar, 20 μm. (B) Quantification of Ki67 immunofluorescence staining in CD73-expressing cells relative to neighbor untransfected cells (30 CD73+ cells and 150 CD73− untransfected cells were counted per condition). ****p < 0.0001, unpaired t test. (C) Measurement of 5′-nucleotidase activity in transfected HEK293T cell lysates. Samples expressing equal protein levels (bottom) were analyzed in triplicate. (D) Treatment of CD73-transfected HEK293T cell lysates with peptide-N-glycosidase F results in similar deglycosylation of both isoforms. (E) Immunofluorescence-based localization of Flag-CD73L or Flag-CD73S in primary mouse hepatocytes. Blue, DAPI; bar, 20 μm. (F) Flag immunoblot of Flag-CD73L– and -CD73S–expressing HEK293T total cell lysates analyzed under reducing (+β-mercaptoethanol, βME) or nonreducing (–βME) conditions, showing that CD73S does not dimerize. (G) Coomassie-stained gel of Flag immunoprecipitates of the nonreduced samples shown in F. Asterisk denotes a unique protein present in CD73S immunoprecipitates, identified by mass spectrometry as calnexin. (H) Biochemical validation that CD73S coimmunoprecipitates with calnexin.
Mentions: To determine whether CD73S and CD73L exhibit functional differences, we transfected each isoform into HepG2 cells and monitored the expression of the cell proliferation marker Ki67. The cells expressing CD73L had a significant decrease in Ki67 staining, whereas CD73S expression did not significantly alter Ki67 expression relative to neighboring untransfected cells (Figure 5, A and B). Therefore CD73L and CD73S exhibit functional differences with respect to their ability to modulate the proliferation potential of cultured human HCC cells.

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