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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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CD73S negatively regulates CD73L expression in a proteasome-dependent manner. (A) Measurement of 5′-nucleotidase activity in lysates from HEK293T cells transfected with equal amount of Flag-CD73L cDNA together with empty vector or Flag-CD73S. ****p < 0.0001, unpaired t test. (B) Measurement of CD73L (endogenous) and CD73S (transfected) mRNA in Huh-7 cells, showing that CD73S does not affect CD73L mRNA. (C) Determination of the effect of increasing levels of CD73S on CD73L dimer formation, which is detected under nonreducing conditions (–βME). (D) CD73 immunoblot of CD73-transfected HEK-293T lysates (±MG132). The same membrane under two exposure times from a triplicate experiment is shown. (E) Quantification of blot from D, showing that MG132 restores CD73L protein levels. ***p < 0.001, unpaired t test. (F) Reciprocal coimmunoprecipitation of CD73S/L-transfected HEK293T lysates, demonstrating that CD73S complexes with CD73L. Anti-V5 antibody was used as a negative control.
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Figure 6: CD73S negatively regulates CD73L expression in a proteasome-dependent manner. (A) Measurement of 5′-nucleotidase activity in lysates from HEK293T cells transfected with equal amount of Flag-CD73L cDNA together with empty vector or Flag-CD73S. ****p < 0.0001, unpaired t test. (B) Measurement of CD73L (endogenous) and CD73S (transfected) mRNA in Huh-7 cells, showing that CD73S does not affect CD73L mRNA. (C) Determination of the effect of increasing levels of CD73S on CD73L dimer formation, which is detected under nonreducing conditions (–βME). (D) CD73 immunoblot of CD73-transfected HEK-293T lysates (±MG132). The same membrane under two exposure times from a triplicate experiment is shown. (E) Quantification of blot from D, showing that MG132 restores CD73L protein levels. ***p < 0.001, unpaired t test. (F) Reciprocal coimmunoprecipitation of CD73S/L-transfected HEK293T lysates, demonstrating that CD73S complexes with CD73L. Anti-V5 antibody was used as a negative control.

Mentions: To assess whether there is cross-regulation between the two human CD73 isoforms, we transfected equal amounts of DNA encoding CD73L in combination with either control vector or CD73S and found that the presence of CD73S caused >50% decrease in the 5′-nucleotidase activity of CD73L, which correlated with decreased CD73L protein expression (Figure 6A). This effect was specific to CD73S, since control overexpression of excess Flag-GAPDH did not result in the same decrease in CD73L protein levels (Supplemental Figure S2B), and the effect was observed regardless of the epitope tag, since GFP-CD73L was similarly down-regulated in the presence of CD73S (Supplemental Figure S2C). Furthermore, the inhibitory effect of CD73S was not limited to the ectonucleotidase CD73L, since CD73S also down-regulated the expression of the cytosolic 5′-nucleotidase IA in a co-over­expression system (Supplemental Figure S2D).


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)

CD73S negatively regulates CD73L expression in a proteasome-dependent manner. (A) Measurement of 5′-nucleotidase activity in lysates from HEK293T cells transfected with equal amount of Flag-CD73L cDNA together with empty vector or Flag-CD73S. ****p < 0.0001, unpaired t test. (B) Measurement of CD73L (endogenous) and CD73S (transfected) mRNA in Huh-7 cells, showing that CD73S does not affect CD73L mRNA. (C) Determination of the effect of increasing levels of CD73S on CD73L dimer formation, which is detected under nonreducing conditions (–βME). (D) CD73 immunoblot of CD73-transfected HEK-293T lysates (±MG132). The same membrane under two exposure times from a triplicate experiment is shown. (E) Quantification of blot from D, showing that MG132 restores CD73L protein levels. ***p < 0.001, unpaired t test. (F) Reciprocal coimmunoprecipitation of CD73S/L-transfected HEK293T lysates, demonstrating that CD73S complexes with CD73L. Anti-V5 antibody was used as a negative control.
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Related In: Results  -  Collection

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Figure 6: CD73S negatively regulates CD73L expression in a proteasome-dependent manner. (A) Measurement of 5′-nucleotidase activity in lysates from HEK293T cells transfected with equal amount of Flag-CD73L cDNA together with empty vector or Flag-CD73S. ****p < 0.0001, unpaired t test. (B) Measurement of CD73L (endogenous) and CD73S (transfected) mRNA in Huh-7 cells, showing that CD73S does not affect CD73L mRNA. (C) Determination of the effect of increasing levels of CD73S on CD73L dimer formation, which is detected under nonreducing conditions (–βME). (D) CD73 immunoblot of CD73-transfected HEK-293T lysates (±MG132). The same membrane under two exposure times from a triplicate experiment is shown. (E) Quantification of blot from D, showing that MG132 restores CD73L protein levels. ***p < 0.001, unpaired t test. (F) Reciprocal coimmunoprecipitation of CD73S/L-transfected HEK293T lysates, demonstrating that CD73S complexes with CD73L. Anti-V5 antibody was used as a negative control.
Mentions: To assess whether there is cross-regulation between the two human CD73 isoforms, we transfected equal amounts of DNA encoding CD73L in combination with either control vector or CD73S and found that the presence of CD73S caused >50% decrease in the 5′-nucleotidase activity of CD73L, which correlated with decreased CD73L protein expression (Figure 6A). This effect was specific to CD73S, since control overexpression of excess Flag-GAPDH did not result in the same decrease in CD73L protein levels (Supplemental Figure S2B), and the effect was observed regardless of the epitope tag, since GFP-CD73L was similarly down-regulated in the presence of CD73S (Supplemental Figure S2C). Furthermore, the inhibitory effect of CD73S was not limited to the ectonucleotidase CD73L, since CD73S also down-regulated the expression of the cytosolic 5′-nucleotidase IA in a co-over­expression system (Supplemental Figure S2D).

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